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A mutational analysis on monoamine oxidase

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A mutational analysis on monoamine oxidase

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Content INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. U M I films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these w ill be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, M l 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. R eproduced with permission of the copyright owner. Further reproduction prohibited without permission. A MUTATIONAL ANALYSIS ON MONOAMINE OXIDASE by Rani Maurice Geha A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL OF PHARMACY UNIVERSITY OF SOUTHERN CALIFORNIA in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (MOLECULAR PHARMACOLOGY AND TOXICOLOGY) May 2002 Copyright 2002 Rani Maurice Geha Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3073779 ___ ® UMI UMI Microform 3073779 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNI VERSI TY PARK LOS ANGELES. CALIFORNIA M O O T This dissertation, w ritten by ....6 j 3L4L ? .____________________ G£U$j& under the direction of h .h .— Dissertation Committee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillm ent of re quirements for die degree of DOCTOR OF PHILOSOPHY Dean of Graduate Studies D ate May...L0-....2 £ > D 2 DISSERTATION COMMITTEE Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rani Maurice Geha Jean Chen Shih ABSTRACT A MUTATIONAL ANALYSIS ON MONOAMINE OXIDASE Little is known about the active site of monoamine oxidase (MAO) and what determines substrate and inhibitor specificities of its two types, MAO A and B. As of yet, the structure of MAO could not be obtained by X-ray diffraction because MAO is a membrane bound enzyme, it has very low solubility and is therefore difficult to crystallize. In this work we produce a soluble form of MAO B by removing 40 amino acids from the C-terminus. This soluble form, termed MAO B-C481, is 48% soluble, compared to 0.3% of wild-type MAO B, and should crystallize more readily. We have used site-directed mutagenesis to better the active site of MAO. Using polyamine oxidase as a model, an enzyme with 20% amino acid identity with MAO, we have identified Lys-305 and Trp-397 in MAO A and their corresponding residues in MAO B, Lys-296 and Trp-388 as forming critical non-covalent bonds with the FAD moiety of MAO. We have also identified an aromatic sandwich structure within the substrate binding site. It consists of the aromatic portion of tyrosines 407 and 444 in MAO A and tyrosines 398 and 435 of MAO B. The amino acids have a very similar function in both subtypes since mutating them produces a very similar effect on MAO A and B. This indicates that the active structures of the two isoenzymes are similar. We have also identified the residues which determine substrate and inhibitor specificities in MAO A and B. When Ile-335 of MAO A and Tyr-326 o f MAO B were reciprocally interchanged, the specificities of mutant MAO A and B were switched. I Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Mutant MAO A-I335Y preferred the MAO B substrate PEA over the MAO A substrate 5- HT and was more sensitive to deprenyi, the MAO B specific inhibitor, than to clorgyline, the MAO A specific inhibitor Similarly, MAO B-Y326I exhibited an increased activity for 5-HT, a decreased activity for PEA and was more sensitive to clorgyline than deprenyl. This indicates that Ile-335 in MAO A and Tyr-326 in MAO B control substrate and inhibitor specificity. 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dedicated to my family DEDICATION Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDMENTS I am highly indebted to my mentor, Jean Shih, for teaching me the skills needed to be an independent researcher. I would also like to thank Kevin Chen for helpful discussions and for always having the answers to my molecular biology questions and Joseph Grimsby for getting me up to speed by teaching me the techniques used in our lab. Much of the work in my dissertation would not have been possible without Igor Rebrin for expressing some of my mutant MAOs in the baculovirus system and for doing the solubility and imaging experiments for the C-terminally truncated MAOs. 11 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS D edication .................................................................................................................................................................... ii A cknow ledgm ents .....................................................................................................................................................iii List o f T ables ................................................................................................................................................................v List o f F ig u re s ............................................................................................................................................................. vi B ackground on M onoam ine O x id a se ....................................................................................................................1 Structure and active s ite ..................................................................... 3 FAD binding............................................................................................................................................ 3 Catalytic s ite ........................................................................................................................................... 4 Substrate specificity............................................................................................................................... 5 I. E ffe c t s o f C arboxy-t e r m in a l T ru n c a t io n s on H uman M o n o a m in e O x id a se B a c t iv it y and So l u b il it y .................................................................................................................................11 Introduction and ratio n al...........................................................................................................................11 R esults ....................... ....14 Activities o f wild-type and C-terminally truncated mutant MAO Bs ................................. 14 Kinetic characterization o f C-terminal truncation mutants.................................. .............../6 Solubility o f C-terminal truncation mutant proteins................ 17 Expression and characterization o f GFP-MAO fusion proteins............................................/9 D iscussion.............................................................. 20 II. Id e n t ific a t io n o f t h e a c t iv e S it e and t h e S pe c if ic it y D e t e r m in in g R esid u es o f M o n o a m in e O xid a se A and B u sin g s it e- d ir ec ted m u t a g e n e s is....................................... 26 1) Five key amino acids play sim ilar roles in the active site o f M A O ................................................. 26 Introduction and ratio n al.......................................................................................................................... 26 R esults.............................................. 32 MAO A mutants............................................. 32 MAO B mutants..................................................................................... 34 D iscussion ................................................................................................................................................ 36 FAD binding site...........................................................................................................................37 Substrate binding site...................................................................................................................38 2) Phe-208 and lie-199 in hum an monoamine oxidase A and B do not determ ine substrate and inhibitor specificities as in r a t ........................................................................................... 41 Introduction and ra tio n a l...................................... 41 R esults................................................................................................................................................ .43 D iscussion....................................................................................................................................................47 3) Substrate and inhibitor specificities in hum an MAO A and B are determ ined by Ile-335 and Tyr-326 respectively.......................................................................................................52 Introduction and ratio n al.......................................................................................................................... 52 R esults..................................................................................................... 54 D iscussion................................................. 63 C o n clu sio n ...................................................................................................................................................................66 E xperim ental P ro c e d u re s ......................................................................................................................................70 References .............................................................................................................................................77 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table 1.— K i n e t i c p r o p e r t i e s o f C - t e r m i n a l t r u n c a t i o n m u t a n t s ....................... 16 Table 2 .— A c t i v i t i e s of MAO A a c t i v e s i t e m u t a n t s ....................................................... 33 T a b l e 3 .— In h ib it io n c o n s t a n t s o f MAO A a n d B a c t iv e s it e m u t a n t s ............. 3 4 Table 4.— A c t i v i t i e s of MAO B a c t i v e s i t e m u t a n t s ....................................................... 36 T a b l e 5 .— A c t i v i t i e s of c h i m e r i c MAOs a n d m u t a n t s A-F208I, B-I199F.......... 43 Table 6.— KM values of chimeric MAOs and mutants A-F208I, B-I199F.......... 4 4 T a b l e 1.— IC5 o v a l u e s of c h i m e r i c MAOs a n d m u t a n t s A-F208I, B-I199F....... 4 5 Table 8.— K cat a n d Km values of A-I335Y a n d B-Y326I......................................56 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES F ig . I .— M u l t i p l e s e q u e n c e a l i g n m e n t of M A O s ................................................................. 6 F ig . 2 .— C ARBOXY-TERMINAL M A O B TRUNCATION MUTANTS.......................................13 F ig . 3 .— A c t iv i t ie s o f C - t e r m i n a l t r u n c a t io n m u t a n t s ..............................................15 F ig . 4 .— S o l u b il it y o f C - t e r m i n a l t r u n c a t io n m u t a n t s ..............................................18 F ig . 5 .— C o m m o n F A D b in d in g a m in o a c id s in M A O a n d P A O ................................2 8 Fig. 6 .— C o m m o n s u b s t r a t e b i n d i n g a m i n o a c i d s in M A O a n d P A O ................. 2 9 F ig . 7 .— T h e F A D binding s it e ........................................................................................................... 3 0 Fig. 8 .— T h e s u b s t r a t e b i n d i n g s i t e ............................................................................................. 31 Fig. 9 .— C o n s t r u c t i o n o f M A O A a n d B p o i n t m u t a n t AND CHIMERIC ENZYMES......................................................................................................... 4 2 Fig. 1 0 .— M u l t i p l e s e q u e n c e a l i g n m e n t o f t h e p u t a t i v e SPECIFICITY DETERMINING 1 6 6 AMINO ACID SEGM ENT......................................... 53 Fig. 1 1 .— K cat/K m values of A -I3 3 5 Y a n d B - Y 3 2 6 I ................................................................5 7 F ig . 12.— C l o r g y l in e a n d d e p r e n y l in h ib it io n c u r v e s o f A -1 3 3 5 Y and B - Y 3 2 6 I ....................................................................................................61 Fig. 1 3 .— Ro 4 1 - 1 0 4 9 a n d Ro 1 6 -6 4 9 1 inhibition curves OF A - I 3 3 5 Y AND B - Y 3 2 6 I ....................................................................................................6 2 Fig. 14.— S c h e m a t i c M A O m o d e l ........................................................................................................ 6 8 F ig . 1 5 .— 3 - d im e n t i o n a l m o d e l o f M A O ....................................................................................... 6 9 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BACKGROUND ON MONOAMINE OXIDASE Monoamine oxidase [MAO; amineroxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4] is an integral outer mitochondrial membrane protein (Greenawalt and Schnaitman, 1970) and catalyzes the oxidative deamination of various biogenic amines in the CNS and peripheral tissues. MAO exists in two isoforms, type A and type B (Johnston, 1968; Squires, 1968). MAO A preferentially oxidizes serotonin (5-HT) and is inhibited by low concentrations of the irreversible inhibitors clorgyline (Johnston, 1968) and by the reversible inhibitors Ro 41-1049 (Cesura et al., 1989), brofaromine and moclobemide (Da Prada et al., 1990). MAO B has a high affinity for P-phenylethylamine (PEA) and is inhibited by low concentrations of deprenyl (Knoll and Magyar 1972), Ro 19-6327 (Da Prada et al., 1990), Ro 16-6491 (Cesura et al., 1988) and MDL-72145 (Bey et al., 1984). Common substrates include tyramine (TA) and dopamine. (For a review of MAO see Shih et al., 1999). MAO plays an important function in the regulation of mood and affect. It was discovered that the inactivation of MAO A via a point mutation resulted in abnormal aggression in a group of males siblings (Brunner et al., 1993). The knocking-out of the MAO A gene in mouse also resulted in increased aggression (Cases et al., 1995). The function of MAO A and B in the brain is to regulate the level of monoamine neurotransmitters, such as serotonin, by degrading them to the corresponding aldehyde after reuptake into the pre-synaptic nerve terminal. This has made MAO an important target for the treatment of mood disorders such as depression 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. using MAO inhibitors (MAOIs). However the usefulness of these compounds was hampered by their tendency to elicit hypertensive crises when MAOIs were ingested with foods rich in tyramine, such as dairy products, leading to the so-called “cheese reaction”. The biochemical cause o f this effect was the inhibition of both subtypes of MAO by poorly selective MAOIs preventing the degradation of the common substrate tyramine and leading to tyramine potentiation. The more recently developed MAOIs with higher selectivity, such as the MAO A inhibitor moclobemide, had a superior safety and efficacy that the classical MAOIs but a low tyramine diet was still needed to reduce the risk of a cheese reaction. The three dimensional structure of MAO and its active site is important for the development of highly selective MAOIs and is not yet available. In the following studies we attempt to elucidate the structure of MAO and its active site in order to allow a structurally based approach for the development of highly selective MAOIs. One of the major impediments for the crystallization of MAO and obtaining its structure via X-ray crystallography is the fact that MAO is a membrane bound protein and therefore difficult to get into solution, a necessary step before crystallization. In Section I, we produce a soluble form of MAO B. The development of specific inhibitors for MAO A and B following a structural approach necessitates an understanding of the active site amino acids and the identification of the residues which determine A and B specificities. In Section II we use site-directed mutagenesis to understand the active site o f MAO A and B, and also to identify the key amino acids responsible for their distinct substrate and inhibitor 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. specificities. For this approach we have made use of the fact that MAO A and B have arisen from an ancestral genetic duplication event (Grimsby et al., 1991). This allows us to study the amino acids important for MAO activity by targeting amino acids that have been conserved throughout evolution and among different species. Amino acids which are important for determining substrate and inhibitor specificities are the ones that are different between MAO A and B but conserved among different species of one subtype. Str u c tu r e a nd activ e site Human MAO A and B consist of 527 and 520 amino acids respectively and have a 70% amino acid identity (Bach et al., 1988). Their repective molecular weights are 59.7 and 58.8 kDa. The amino acid identity of one MAO isoenzyme across different mammalian species is 87-88% (Chen et al., 1994). The primary sequence alignment of MAO from various species (Fig. I) reveals three highly conserved regions and a hydropathy plot shows six hydrophobic regions (Bach et al., 1988). a) FAD binding Each MAO molecule contains one covalently attached FAD coenzyme. It was identified by the isolation o f a pentapeptide, Ser-Gly-Gly-Cys(FAD)-Tyr, containing FAD linked at the cysteine residue (Cys-406 in MAO A, Cys-397 in MAO B) site via a 8a-methyl-S-cysteinyl linkage to the FAD isoalloxazine ring (Kearney et al., 1971, 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Walker et al., 1971). Mutation of this cysteine residue to serine results in the deactivation of the enzyme (Wu et al., 1993). In addition to the covalent cysteine attachment site, four amino acids have been identified in MAO B as having a noncovalent attachment function to FAD. These four amino acids (Glu-34, Arg-42, Tyr-44, and Thr-45) are highly conserved between MAO and other flavoproteins and were shown to inactivate the enzyme when mutated (Kwan et al., 1995, Zhou et al., 1995“ ' b, Kirskey et al., 1998). They are part of a 50 amino acid FAD binding motif and highly conserved among a superfamily of 29 flavoproteins to which MAO belongs (Dailey and Dailey, 1998) and located near the N-terminus. A dominant feature of this binding motif is a p-sheet-a-helix-P-sheet structure (Pi-a-Pi). However the homology of this superfamily does not extend beyond this motif. For this reason, our active site studies have focus on the region outside of this motif and specific to MAO. b) Catalytic site Little is known about the catalytic site of MAO. Although it is assumed that the substrate needs to bind close to the three ringed isoalloxazine moiety of FAD, the oxidoreductive center of the enzyme, amino acids mutations that dirupt activity have been shown to be due to the disruption of FAD binding and not substrate binding or catalysis. One amino acid however, Cys-365 in MAO B has been shown to form a covalent adduct with the antidepressant fra/ur-2-Phenylcyclopropylamine (Zhong and 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Silverman, 1997). This represents the only amino acid shown to belong in the active site and is not associated with FAD binding. c) Substrate specificity There has been interest in identifying the residues that determine the substrate and inhibitor specificities of MAO A and B because they will aid in the development of highly specific MAOIs. Lacking any information on the nature of the substrate binding pocket, several groups have approached this problem through the construction of chimeric MAO A and B molecules (Gottowik et al., 1993, 1995, Tsugeno et al., 1995, Grimsby et al., 1996, and Chen et al., 1996). These experiments consisted of reciprocally interchanging corresponding regions of MAO A and B with the objective of switching their substrate and inhibitor specificities. Some chimerics were inactive, implying some structural incompatibilities between A and B, while others showed only a partial change in specificity. Although this approach was unable to produce a clean specificity switch, it nevertheless provided a useful guide as to where the specificity determining residues might be. Also, active chimerics that did not show any change in specificity when compared to their parent enzyme were useful in excluding the N-terminal and the C-terminal portions as being important for specificity (Gottowik et al., 1993, and Chen et al., 1996). A chimeric study involving the middle segment of human MAO suggested that specificity may be determined within amino acid segment 161-375 in MAO A and its corresponding segment in MAO B, 152-366 (Grimsby et al., 1996). 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1. Multiple sequence alignment o f MAOs. MAO A from mouse, rat, bovine, and human, and MAO B from mouse, rat, and human, and trout MAO are aligned. The common N-terminal FAD binding motif of a flavoprotein superfamily is underlined. Highly conserved regions are shaded. The cysteine at which FAD covalently attaches is marked by an arrow undemeath“T”. M-MAO A 1 R-MAO A 1 B-MAO A 1 H-MAO A 1 M-MAO B 1 R-MAO B 1 H-MAO B 1 T-MAO 1 MTDLEKP S I TGHMFDVGLIGGGTSGIiAJflUCLL'SE L » • • • * • MT.A • • • • > • ' • " * - ' • • •*- • »• •* • i i r v i • • • «. « « ^ • i • • « •- : ES :ENQ: MSNKS; :3THD MTAQNT 34 34 34 34 2 5 25 25 27 M-MAO A 35 R-MAO A 35 B-MAO A 35 H-MAO A 35 M-MAO B 2 6 R-MAO B 26 H-MAO B 26 T-MAO 28 KN: 68 68 68 68 59 59 59 6 1 M-MAO A 69 R-MAO A 69 B-MAO A 69 H-MAO A 69 M-MAO B 60 R-MAO B 60 H-MAO B 60 T-MAO 62 :H: ::::::::::::::S ::::: : :A : : : : L : : : : : : EV: : : IHF :A : : : : L : : : : : : EV: : : IHF :A : : : : L : : : : : : EV: : : IHH 62 : : : : : : : : : : : : A : :C:V K : I : : : EE: : : :H: 102 102 102 93 93 93 95 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1 (continued). M-MAO A 1 0 3 GKTYPFRGAFPPVWNPLAYLDYNNLWRTMDDMGK 13 6 : 1 3 6 : 1 3 6 : 1 3 6 M-MAO A 103 R-MAO A 1 03 B-MAO A 1 0 3 H-MAO A 1 0 3 M-MAO B 94 R-MAO B 94 H-MAO B 94 T-MAO 96 S : A : S : A : S : : : P: P: P: J • • • • • • • • • • • • • • • J ••••••••••• J • • I T : : :N: : : : : : : :EM:Q 127 127 I T : : : H: : F : : : : : : : : R 127 S : : : K : S : : :M:: :F:LM: : : : : : : K: : E : : S 129 M-MAO A 1 3 7 EIPVDAPWQARHAEEWDKITMKDLIDKICWTKTA 1 7 0 M-MAO A 1 3 7 R-MAO A 13 7 B-MAO A 1 3 7 H-MAO A 13 7 M-MAO B 12 8 R-MAO B 12 8 H-MAO B 12 8 T-MAO 13 0 :E:P: :Q: : V : : M : : M : • • • • • • • • • • • • ^ Q : : : E : : : : : : : : 170 :T : : : : E :Q: : DK: : : M: : : E : : : : : : : : : : : 170 :S : : : :K:PL:: : : :YM:: : E : L : : : : : :NST 161 ;S : : : : K:PL: : : : : YM: : : E :L : : : : : :NST 161 : S : : : : K: PL : : : : :NM : : : E : L : : L : : : ES : 161 :RE: : : K : P : : : : : : :M:: : Q : F : : : : : :SS: 163 M-MAO A 1 7 1 RECAY R-MAO A 1 7 1 : : F : : B-MAO A 1 7 1 : QF : S H-MAO A 1 7 1 : RF : : M-MAO B 1 6 2 KQI : T R-MAO B 1 6 2 KQI :T H-MAO B 1 6 2 KQL :T T-MAO 1 6 4 : R F : T vXV-T r . 1 Jy 1» v i* . . : • . * ; % ; ► ! • • T , f ™ * • • A * . . . . — - - > •!">« - V - 1 '.* T^"-V ‘r- " • •• •• • * * * * * * s i" - 2 0 4 2 0 4 2 0 4 2 0 4 1 9 5 1 9 5 1 9 5 1 9 7 M-MAO A 2 0 5 R-MAO A 2 0 5 B-MAO A 2 0 5 H-MAO A 2 0 5 M-MAO B 1 9 6 R-MAO B 1 9 6 H-MAO B 1 9 6 T-MAO 1 9 8 • ■ I- •-T 5 \ " * £ ' ^tO;’ S j •? • v E • _ • ■ • ' . .. ■ .. • . • y ,- l - J ' ■ -u'-'<.'<V • i ': : .v '.t i L" • ■y • > •i'* r V -I•,:.•»*V ; * ~ i ::Q: 238 : : R : 22 9 : : R: 229 : : R: 2 2 9 :ER: 2 3 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1 (continued). M-MAO A 2 3 9 KLS S PVTYIDQTDDNIIIETLNHEHYECKYVISA R-MAO A 2 3 9 : : ; : : : : : : : : V : : : : : : ; B-MAO A 2 3 9 : R: : : V SSE : T V : R L : R : : H-MAO A 2 3 9 : NH :HV SS : I I I I ; z N: M-MAO B 2 3 0 : ER IH : : GE V : VK I A : R-MAO B 2 3 0 : ER IH : :GE V W K I AK H-MAO B 2 3 0 : ER I : : : RE VLV: M A : T-MAO 2 3 2 : ME : YK: : G :MVEV: K T KA: V: 2 7 2 2 7 2 2 7 2 2 6 3 2 6 3 2 6 3 2 6 5 M-MAO A 2 7 3 IPPVLTAKIHFKPELPPERNQLIQRLPMGAVIKC 3 0 6 M-MAO A 2 7 3 R-MAO A 2 7 3 B-MAO A 2 7 3 H-MAO A 2 7 3 M-MAO B 2 6 4 R-MAO B 2 6 4 H-MAO B 2 6 4 T-MAO 2 6 6 GM GM GM NL M R R YSAP HS : P :N:P :N: : S A ML IL MM :L M S V L S T V L S T V L s H V : s 306 306 306 297 297 297 299 M-MAO A 3 07 MVYYKEAFWKKKDYCGCMIIEDEEAPISITLDDT 3 4 0 : : : : : : : : : : : : : : : : : : : : : : : : : :A: : : : : : 340 M: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 340 M: : : : : : : : : : : : : : : : : : : : : D : : : : : : : : : : 340 :R : P : : R : : : F : : T : V : : G : : : : : AY: : : : : 331 : : : P : : R :: : F : : T : V : : G : : : : : AY: : : : : 331 I : : : : : P : : R : : : : : : T : : :DG:: : :VAY:: : : : 331 I : : : R : N : : R : : G : : : T : V : : E : : : : :GL:: : : : 33 3 M-MAO A 3 0 7 R-MAO A 3 0 7 B-MAO A 3 0 7 H-MAO A 3 0 7 M-MAO B 2 9 8 R-MAO B 2 9 8 H-MAO B 2 9 8 T-MAO 3 0 0 M-MAO A 3 4 1 KPDGSMPAIMGFILARKAERLAKLHKDIRKRKIC 3 7 4 R-MAO A 3 4 1 : :L : P i ! I ! * ! * i ! ! * ! * ! ■ 3 7 4 B-MAO A 3 4 1 : :L : D : : : : V : : : : : : : : : : 374 H-MAO A 3 4 1 : :L : D: : : : : : :E : : :K: : : 3 7 4 M-MAO B 3 3 2 :TYA H: RK: VR:T:EE:L : :L: 365 R-MAO B 3 3 2 AGCA H: RK:V R : T : E E : L : :L : 3 6 5 H-MAO B 3 3 2 E :NYA H: R K : : R : T : E E : L K :L : 3 6 5 T-MAO 3 3 4 : TV t : : CRK: CG: T : E E : : K R :: 3 6 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1 (continued'). M-MAO A 3 7 5 R-MAO A 3 7 5 B-MAO A 3 7 5 H-MAO A 3 7 5 M-MAO B 3 6 6 R-MAO B 3 6 6 H-MAO B 3 6 6 T-MAO 3 6 8 M-MAO A 4 0 9 R-MAO A 4 0 9 B-MAO A 4 0 9 H-MAO A 4 0 9 M-MAO B 4 0 0 R-MAO B 4 0 0 H-MAO B 4 0 0 T-MAO 4 0 2 M-MAO A 4 4 3 R-MAO A 4 4 3 B-MAO A 4 4 3 H-MAO A 4 4 3 M-MAO B 4 3 4 R-MAO B 4 3 4 H-MAO B 4 3 4 T-MAO 4 3 6 : H i: : H :* : 5 » j;^ 3 3 & s R ii-fc « « = 5 ' GYMEGAVEAGERAAREVLNALGKVAKKDIWVQEP ‘ c > » •■•-», » • ‘ • • • • • • • • • • • • • • L* • • • -•.#• *. • • • • • • • • • • • • • • • X.J • • * •■ ••- » • • • • • • • C A • • • • T O • • * • » • • • • • • • UrV • • • • J > \ / • • r > rrr? • : •■■ * ■•-■ • '*-T' ^*-1 • * ( i * • * • I n * * * * * * * • ■ - " • • w • • » • X U • • • • • • • • ^ . ~ ^ i t l j : H : I : :IPEDE:W Q P: : : H : I : : IPEDE : : QP : : : i : H : M: : IPEDE : : QS : : i.V 'v t i'i^ S S ^ S S lM Y E M : R IPQ SQ s :QT: : 4 7 6 4 7 6 4 7 6 4 7 6 4 6 7 4 6 7 4 6 7 4 6 9 M-MAO A 4 7 7 ESKDVPALEITHTFLERNLPSVPGLL R-MAO A 4 7 7 ::::: I : : : : : : : : : : : : : : : : : : B-MAO A 4 7 7 • AT?* • • • \T • • • D Q • W • • • • • • • Q • • * • riu • • • • v » • « & m • t i • • • * • • • w • • • H-MAO A 4 7 7 • • • • • » •••••• ••••••• ^ • • • M-MAO B 4 6 8 • ^ • • • • p • • 2 • • • • • pj •••*•••• R-MAO B 4 6 8 • ^ • • • • ^p • • » • • • • »••••••• H-MAO B 4 6 8 • ^ • • • • q p • • *p • • • • • pj •••••••• T-MAO 4 7 0 VE: : : : PFVT: : W: : : : : : : KITGFS 5 0 8 : : : :V: :: V : :: 508 508 508 491 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1 (continued). M-MAO A 5 0 9 TSVALLCFVLYKFKQPQS 5 2 6 R-MAO A 5 0 9 : : : : : : : : : : : : I :KLPC 5 2 6 B-MAO A 5 0 9 : : IT A :W : : M: RFRL: SRS 5 2 7 H-MAO A 5 0 9 : : : T : : G: : : : : Y :LLPRS 5 2 7 M-MAO B 5 0 2 L :A T A :G : L P T :GAVCTFLKMGFRA 5 2 6 R-MAO B 5 0 2 L :A T A :G : LAH: KGLFVRF 5 2 0 H-MAO B 5 0 2 F :A T A :G : LAH: RGLLVRV 5 2 0 T-MAO 4 9 2 G G FIN : LA 4 9 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Section I E ffects o f C a rbo xy-ter m in a l T r u n c a t io n s o n H u m an M o n o a m in e O xidase B A c t iv it y a n d So l u b il it y INTRODUCTION AND RATIONAL One of the major impediments to obtaining the X-ray crystal structure of MAO is that it is a membrane-bound protein. Unlike soluble proteins which have a hydrophobic core and a hydrophilic surface, membrane bound proteins have a large exposed hydrophobic portion making them especially difficult to get into solution, a necessary step before crystallization. In this study we attempt to solubilize MAO B by exploiting a presumed feature of the protein: Attachment to the membrane through a single C- terminal hydrophobic a-helix. Although MAO is an integral outer mitochondrial membrane protein (Greenwalt and Schnaitman, 1970) it lacks the common N-terminal hydrophobic region flanked by positively charged sequences typical of the large group of outer mitochondrial anchored proteins (Shore et al., 1995) and its insertion into the outer mitochondrial membrane is ATP and ubiquitin dependent (Zhuang et al., 1992). Deletion of the 55 N-terminal amino acids in rat MAO B has no effect on the targeting or the insertion of the enzyme into the outer mitochondrial membrane after expression in mammalian cells (Mitoma and Ito, 1992). The mitochondrial targeting signal of rat liver MAO B was shown to be within the carboxy terminal residues 492 to 520 by fusion of this segment to the C- terminus of cytochrome bs which directed this chimeric protein to the outer 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mitochondrial membrane. However, a mutant of human MAO A lacking the last 24 C- terminal amino acid residues was still found in the membrane fraction (Weyler et al., 1994). The cloning of the flavin-containing polyamine oxidase from maize (Tavladoraki et al., 1998) revealed a 20% sequence identity with MAO. This enzyme, which is soluble, is missing a 50 carboxy-terminal amino acid stretch present in MAO. This further indicates a possible C-terminal anchoring of MAO to the membrane. The removal of the C-terminal anchor could therefore allow the solubilization of MAO without having an effect on the overall structure of the protein. In this study we examine the role of the 123 carboxy-terminal residues spanning from amino acid positions 397 to 520 of human MAO B and attempt to create a soluble form of the enzyme that would be more suitable for crystallization. We have introduced stop codons by site-directed mutagenesis to generate 10 progressive C-terminal truncation mutants (Fig. 2). The mutants were expressed in Sf21 insect cells following transfection with baculovirus DNA containing the mutant MAO B cDNA and were analyzed for activity and solubility. We show that MAO B can be solubilized by the removal of 40 C-terminal amino acids without disrupting the active site. 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2. Carboxy-terminal MAO B truncation mutants. In the upper part is the schematic representation of MAO B. Shaded boxes represent six hydrophobic regions (I-VI) identified by hydropathy plot (Bach et al., 1988). Clusters of two or three consecutive positively charged amino acids flanking this regions are indicated on top. In the bottom, the C-terminal amino acid sequence for MAO B wild-type and all 10 truncation mutants starting with residue 396 arc shown. Letters in bold indicate positively charged amino acid residues. The putative hydrophobic transmembrane regions is underlined, it and H it indicate single or double stop codons. The cysteine residue where FAD covalently attaches is marked with an arrow ”4 - ”. — indicates a break in the sequence. f a d RKK , RH HKR it / / 1(6-22) 11(101-121) 111(161-191) I V (286-306) V (3 9 |-4 4 |) VI(476-5ao) / ✓ ✓ 4 4 4 4 4 4 4 400 4 1 0 420 470 4 8 0 4 9 0 500 510 520 I I I II I I I I I MAO-B GCYTAYFPPGILTQYGRVLRQPVDR-- -EPESVDVPAQPITTTFLERHLPSVPGLNRLIGLTTIFSATALGFLAHKRGLLVRV C 511 GCYTAYFPPGILTQYGRVLRQPVDR EPESVDVPAQPITTTFLERHLPSVPGLNRLIGLTTIFSATALGFL# C 504 GCYTAYFPPGILTQYGRVLRQPVDR-- -EPESVDVPAOPITTTFLERHLPSVPGLNRLIGLTTIFS# C 498 GCYTAYFPPGILTQYGRVLRQPVDR-- -EPESVDVPAOPITTTFLERHLPSVPGLNRLIG# C492 GCYTAYFPPGILTQYGRVLRQPVDR-- -EPESVDVPAQPITTTFLERHLPSVPG# C486 GCYTAYFPPGILTQYGRVLRQPVDR-- -EPESVDVPAQPITTTFLERHL# C 481 GCYTAYFPPGILTQYGRVLRQPVDR-- -EPESVDVPAQPITTTF## ~ C476 GCYTAYFPPGILTQYGRVLRQPVDR-- -EPESVDVPAQ## C467 GCYTAYFPPGILTQYGRVLRQPVDR-- -E# C417 GCYTAYFPPGILTQYGRVLRQ# C397 G## u > RESULTS Small-scale expression was conducted in adherent cell cultures of S£21 cells for the 10 C-terminal truncation mutants and full-length wild-type MAO B. Cells were then harvested, washed, lysed, and the crude homogenates, which showed the appearance of a major band with a Mr of 60 kDa compared to mock infected cells on the SDS gel, were used for activity, kinetic, and solubility studies. Activities of wild-type and C-terminally truncated mutant MAO Bs Specific activities using the substrate phenylethylamine of C-terminally truncated mutant proteins compared to wild type MAO B are shown in Figure 3. Activity gradual decreases the larger the size of the truncation until it reaches complete inactivity for C397. Inactivity for C397 is expected since the FAD cofactor attaches at Cys-397. The level of expression for all mutant proteins was approximately the same as full-length wild type MAO B as demonstrated by immunoblot analysis using polyclonal anti human MAO B antibody. No trace of full length MAO B could be detected in the cell homogenates of C-terminal truncated mutants by western blot analysis. 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3. Activities o f C-terminal truncation mutants compared to full-length human MAO B. Specific activities of Sf21 cell homogenates were measured with dopamine as substrate. The values are represented as percentage of wild type MAO B activity. Data are means ± SEM of three experiments. 12 10 M AO B C 511 C 504 C498 C 492 C 4 8 6 C 481 C 476 C 467 C 417 truncation mutants 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Kinetic characterization of C-terminal truncation mutants Table I shows the kinetic properties of the C-terminal truncated mutant proteins using PEA as a substrate and clorgyline and deprenyl as inhibitors. A decrease of up to 4 fold in V m ax for PEA was found in the mutants up to position 498. Further truncations lead to a 10-100 fold drop in V m aX values. However the Km for PEA was found to be almost the same for all C-terminal truncated mutant proteins as for the full length MAO B indicating that the truncations did not have an effect on affinity. A slight increase in IC50 (representing a decrease in sensitivity) for deprenyl and clorgyline was observed. Table 1. Kinetic properties o f C-terminal truncation mutants. Kinetic parameters and IC50 values of wild-type and carboxy-terminal truncation mutants of MAO B expressed in Sf2l cells. Km and Vmax values of the wild-type and carboxy-terminal truncation mutants were determined as described in Materials and Methods. Vmax is expressed as nanomoles per min per milligram of protein. Data are means ± SEM of three experiments. PEA Deprenyl Clorgyline Enzyme Km ____________ V™ *_______ IC50 (10'9 M) ICS 0 (10~7 M) MAO B 1.54 + 0 . 6 8 .5 2 + 0 .1 7 3 .8 + 0 .0 8 5 . 6 ± 1 .1 C511 1 .1 6 + 0.0 8 9 .6 0 + 2 . 9 3.7 ± 0 .3 5 4 . 7 0 .0 6 C504 1.50 ± 0 .66 4 .4 0 ± 0 .26 4.3 ± 0.0 8 7 . 6 ± 2 .14 C498 2 .0 5 ± 0 .9 5 2 . 0 1 + 0 .24 6 .7 + 1 .6 7 .4 1 .9 C492 1.41 ± 0.33 0 .43 + 0.07 6 . 6 + 0 .57 6 . 9 ± 0 .14 C486 1 .8 6 ± 0 .5 1 0 .11 + 0 .017 6.8 + 2 . 1 1 2 .7 + 2 .4 C481 2 .15 ± 0 .5 2 0 .054 ± 0.02 19 .7 3 .15 1 5 .2 ± 0 .13 C476 2 .20 ± 0 .55 0 .0 5 ± 0 .01 20 .4 ± 4 . 3 5 14 .3 + 5.0 C467 1.53 ± 0 .5 9 0 .2 9 ± 0 .09 7 .9 ± 0.8 7 . 4 0 .9 C417 1.73 ± 0.33 0 .0 4 ± 0 .03 4 .9 ± 2 . 1 4 .8 ± 3.8 C397 - - - - 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Solubility of C-terminal truncation mutant proteins The homogenates of S£21 insect cells expressing the C-terminal truncated mutants were subjected to ultracentrifugation at 100,000 x g for 60 min. The homogenates and supernatants were then separated and MAO B activity was measued in each fraction. The activities of MAO B retained in S100 extracts are summarized in Figure 4. The full length MAO B and mutants C511, C504, C498 were completely sedimented during ultracentrifugation as demonstrated by the lack of significant activity found in the SI00 fraction. C-terminal truncated mutants C492, C486 and C481 show increased “leakage” into the S100 soluble fraction with 10.1%, 22.2% and 47.1% of activity found in the crude homogenates, respectively. However, this pattern did not continue for further truncations as activities of truncation mutants C476, C467 and C417 were predominantly found in the membrane fraction. The retention of the C481 truncated protein in SI00 extract was further analyzed by gel electrophoresis and immunobloting. Full length MAO B was tightly associated with membrane fraction whereas C481 protein after ultracentrifugation was found distributed evenly in the SI00 extract and membrane fraction. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4. Solubility o f C-terminal truncation mutants. Analysis of the solubility of MAO B and its C-terminal truncation mutants in the absence of any detergents. (A) % of total activity of C-terminal truncated mutants and wt MAO B retained in the soluble S i00 fraction after ultracentrifugation o f cell homogenates at 100,000 x g for 60 min. (B) SDS gel electrophoresis of the homogenate (H), S100, and pellet fraction (P) for MAO B, C481, and SOI mock transfected cells. Data are means ± SEM of three experiments. 60 50 4 0 p H 20 • H 10 C492 C486 C481 MAO B C511 C504 C4 98 C476 C467 C417 truncation mutants 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Expression and characterization of GFP-MAO fusion proteins To assess the effect of the truncation of C481 on the intracellular localization of the mutant, we constructed fusion proteins of full length and C481 truncated human MAO B containing Green Fluorescent Protein fused to the amino terminus. After expression in Sf2l, live insect cells were analyzed by fluorescent microscopy. GFP-MAO B fusion protein was found to be expressed in the mitochondria which appears as distinct spots of fluorescence of the cell. In contrast, cells expressing GFP-C481 fusion protein were found throughout the cytoplasm which appears as confluent fluorescence within the cell. 44% of activity of the GFP-C481 mutant was retained in the soluble S100 fraction. 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DISCUSSION It has been proposed that the C-terminal 30 amino acid residues of mitochondrial MAO contribute to the targeting and membrane anchoring of this enzyme to the outer mitochondrial membrane. In one study, a chimeric construct of the soluble cytochrome b5 with the 492-520 peptide from rat MAO-B was targeted to mitochondria in COS-1 cells (Mitoma and Ito, 1992). In another study, truncation of the 24 C-terminal residues of human MAO A did not change the outer mitochondrial localization of this mutant protein after expression in yeast (Weyler et al., 1994). Trout MAO is significantly shorter that other MAOs mostly because it lacks a corresponding carboxy-terminal (Fig. I). Yet trout MAO is bound to the mitochondria like other MAOs. This may indicate an involvement of other regions in membrane targeting and insertion (Chen et al., 1994). The recent interest in the structural basis of the interaction of MAO with the outer mitochondrial membrane triggered our attempt to construct a water soluble form of the MAO B protein by gradual truncations of the C-terminal end, the presumed putative membrane anchor. Expression of such an enzyme, which will not require detergents for extraction and purification, would be beneficial for the crystallization of MAO. The present study used for the first time baculovirus mediated expression of human MAO in insect cells to extend these earlier suggestions and investigate the effects of further truncations on the enzyme kinetic parameters, solubility and intracellular localization. 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Progressive truncation mutants up to position 397 were expressed along with full length MAO B. Mutant C397 was expressed to the same level as the full length enzyme and was completely inactive. This observation is consistent with the result of Wu et al. (1993) in which a Cys397-Ala mutation completely abolished enzyme activity. The absence of measurable activity of C397 mutant suggests that FAD was not incorporated in this protein. However, complete loss of FAD integration in C397 mutant might be an indication on the essential role of the C-terminal portion 397-417 of MAO B for the structural support of FAD docking, since the C417 mutant was active and display Km and IC50 values similar to those of full length enzyme. A progressive and significant decrease in activity correlated with the progression of truncation up to position 417. The decrease of the Vmax for PEA was completely independent from the changes in Km, which was almost the same for all C-terminal truncation mutant proteins. The IC50 towards the irreversible inhibitors deprenyl and clorgyline also reveal only minor changes in all mutants. This indicates that the truncations had a significant effect on the catalytic activity of the enzyme however the fact that substrate affinity and inhibitor sensitivities were preserved indicates that the C- terminal portion of MAO B is not contributing crucial residue(s) to the active site. The large decrease in the reaction rates is also unlikely to be due to the removal of amino acid(s) participating in catalysis in which case activity would be expected to be completely abolished. For example, the C397 mutant lacking covalently attached FAD was completely inactive in our assay conditions, which would allow us to detect enzyme activity 4-5 orders of magnitude lower then those of the wild type MAO B. 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Also, mutating an amino acid known to be involved in catalysis Cys-365 in MAO B (Wu et al., 1993), completely abolishes enzyme activity. The decrease in activity more likely reflects some perturbations of the structure of MAO upon the removal of large C- terminal portion of protein which plays an important role in the interaction with outer mitochondrial membrane. We have also observed that the most soluble C-terminal truncation mutant, C481, became very unstable and rapidly lost activity after separation from the membrane fraction (half-life of about 3 hours). This provides an additional indication on the crucial contribution of the membrane environment to the structural integrity of native MAO. Similar conclusions were drawn from the study on MAO A after truncation of the 24 C-terminal amino acid residues (Weyler et al. 1994). We have also constructed a mutant of human MAO A truncated at the corresponding position of C481. However, unlike MAO B-C481, analysis of this protein reveals complete loss of catalytic activity and unchanged mitochondrial localization after expression of GFP-MAO A(C490) fusion protein in Sf21 insect cells. The different impact o f truncations at position 481 on the catalytic properties of MAO A and MAO B mutant proteins as well as their different intracellular targeting suggest that the structural elements involved in the interaction with outer mitochondrial membrane are not identical, but rather unique for each isoenzyme. The loss of activity of MAO A but not MAO B after truncation at position 481 point towards a crucial role of the C-terminal region for catalytic activity of MAO A but not MAO B. This is in good agreement with previous work on the characterization of chimeric human MAO A/B enzymes (Chen et al., 1996) where it was shown that replacing the 128 C-terminal 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. amino acids of MAO B with the corresponding 126 amino acids from C terminus of MAO-A results in nondetectable activity for the chimeric enzyme. The reverse chimeric, however, was active and exhibited enzymatic properties similar to MAO A wild-type. The effect of truncations on the solubility of MAO B was found to be correlated with the C-terminal 37 amino acid segment (R+ H+ LPSVPGLLR+ LIGLTTIFSATAL GFLAHT K+R+ GLLVR+ V). This segment contains three highly hydrophobic regions, intervened by positively charged amino acids (See Figure 2 for details). Our results indicate that only the complete removal of this region results in maximal solubility. Increased solubility of the C481 mutant as a result of altered intracellular localization was also demonstrated by the expression of GFP-C481 fusion protein. The distribution of fluorescence of this fusion protein throughout the cytoplasm indicates that the protein has lost the targeting signal and has not inserted into the outer mitochondrial membrane. The fusion of the water soluble GFP protein to the N- terminal end of C481 mutant of MAO B, however, has not improved the solubility and/or stability of this protein compared to C481 mutant alone. A more extensive search of conditions, preventing the inactivation of C481, would be necessary to attempt large-scale purification of this protein for crystallization trials. Surprisingly, truncations beyond position 481 resulted in the association with the membrane again (C476, C467, C417). This suggests that the interaction of MAO B with the outer mitochondrial membrane is more complex than a simple anchoring function for the C-terminal 37 amino acids. 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The unexpected solubility behavior of C476, C467, and C417 can be explained as follows, however, unequivocal proof will require further experimental approaches and especially determination of the three-dimensional structure of this membrane protein: Large truncations, such as the removal of up to 24% of the protein as we demonstrated in our present work, can expose the inner hydrophobic region(s) of MAO B thus contributing to a stronger hydrophobic interaction with the mitochondrial membrane. The sequence of MAO B contains six hydrophobic regions, which can be potentially responsible for interaction with membrane (Bach et al., 1988). Segments IV (286-306) and VI (476-520) are flanked by clusters of positively charged amino acids whereas the rest are not. These segments have structural similarity (stretches of hydrophobic amino acids flanked by positively charged ones) to the 22 amino acids long C-terminal transmembrane domain of Bcl-2 (iC’ TLLS L AL V G AC IT LG A YLGH^K*) which has been shown to direct the insertion of this protein into the outer mitochondrial membrane (Nguyen et al., 1993). The different lengths of the above mentioned segments of MAO indicates that this regions might represent p-barrels rather than a bilayer spanning a-helix of 20-22 amino acids as in case for Bcl-2 (Shore et al., 1995). Interaction of MAO B with the outer mitochondrial membrane may also occur through multiple segments of protein, some of them inserted into the membrane and others only interacting with it. The hexameric (trimer of dimers) globular form of bovine liver MAO B found in electron microscopic studies (Shiloff et al., 1996) support the last hypothesis, indicating that a considerable portion of the MAO B protein may be in contact with the mitochondrial membrane. 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In summary, this study shows the construction of a soluble form of MAO and contributes to better understanding the interaction of the MAO B C-terminus with the outer mitochondrial membrane and the role of this domain on the catalytic properties of the enzyme. 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Section II Id e n t ific a t io n o f the A c tiv e S ite a nd th e S pe c ific it y D e t er m in in g R esid u es o f M o n o a m in e O x id a se A a n d B 1) Five key amino acids play similar roles in the active site of MAO INTRODUCTION AND RATIONAL Polyamine oxidase (PAO) catalyses the oxidation of the secondary amino group of polyamines, such as spermine and spermidine. Polyamines are DNA binding molecules which play an important role in cell growth and development (Tabor and Tabor, 1984, Smith, 1985, and Tiburcio et al., 1997). The role of PAO in polyamine catabolism made it an important drug target since some polyamine analogs have been shown to have an antitumoral effect in some cell lines (Pegg and Hu, 1995). It is a monomeric, soluble, 500 amino acid protein with a molecular weight of about 53 kDa and contains a non- covalently bound FAD as a co-factor. It has been classified along with MAO as part of a flavoprotein superfamily having a common 50 amino acid FAD-binding signature motif near the N-terminus (Dailey and Dailey, 1998). PAO shares a 20% amino acid identity with MAO and catalyzes the same chemical reaction: an oxidation half reaction of an amine to an imine coupled to the reduction of O2 to H2O2. These properties suggest that MAO and PAO have similar FAD and substrate binding sites. Recently, the X-ray crystal structure of PAO has been obtained (Binda et al., 1999). We have used the PAO structure as a guide to study the FAD binding amino acids and 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the substrate binding site of MAO. We have aligned amino acids involved with FAD binding and the active site of PAO with MAO A and B from several species to identify conserved amino acids (Fig. 5 and 6). Only conserved amino acids which, according to the PAO structure, mediate their function through their sidechain, and not their mainchain, atoms have been mutated. This criteria allowed us to identify two amino acids, a lysine and a tryptophan (Lys- 305 and Trp-397 in MAO A and Lys-296 and Trp-388 in MAO B), which may play an important role in the noncovalent FAD attachment to MAO (Fig. 7). These residues have been mutated to alanine and were named A-K305A, A-W397A, B-K296A, and B- W388A respectively. We have also identified a structural feature of the substrate binding site of MAO. It is an aromatic sandwich which consists of the sidechains of two parallel tyrosine residues facing the substrate binding pocket on opposite sides (Fig. 8). We have mutated these tyrosines to both phenylalanine and to serine in MAO A and B in order to ascertain the validity of this aromatic sandwich structure. A mutation to phenylalanine is equivalent to the removal of the hydroxyl group from tyrosine while a mutation to serine represents the removal of the aromatic portion. An amino acid associated with the active site of PAO but whose function is not well defined, Glu-120, is conserved in all MAOs. We have mutated this amino acid to alanine in MAO A and B. The mutants were expressed and their activities and sensitivities towards inhibitors determined. 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5. C ommon FAD binding amino acids in MAO and PAO. The function of the fifteen amino acids in PAO associated with FAD binding and their corresponding ones in MAO A and B across various MAO types is shown. Trout MAO (T), human (h-A), rat (r-A), mouse (m-A), and bovine (b-A) MAO A, human (h-B), rat (r-B), and mouse (m-B) MAO B. Identical amino acids are represented by an equal sign“=” Amino acids selected for mutagenesis are highlighted. PAO T h-A r-A m-A b-A h-B r-B m-B Function in polyamine oxidase AA pos. Mainchain N and side chain OH compensate for pyrophosphate negative charge Ser 15 Interacts w/ OH group of ribose Glu 35 Mainchain NH makes H-bond w/ adenine Ala 36 Compensates for pyrophosphate negative charge Arg 43 Carbonyl oxygen bridged by water molecule to N5 of isoalloxazine Gly 57 = = = = = = = = Mainchain NH makes H-bond w/ pyrimidine Asn 59 Ala Ala Ala Ala Ala Ser Ser Ser Mainchain NH and carbonyl oxygen make H-bond w/ pyrimidine Trp 60 Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Mainchain atoms H-bond w/ N1 and N6 of adenine ring Val 237 TLys i S B Nr = v riij .€tB;€§ ^•Trp f$393 tern svr $ $ mm wm li =£• Interacts w1 OH group of ribose Tyr 399 Ser Ser Ser Ser Ser Ser Ser Ser Van der Waals interactions w/ flavin dimethylbenzene ring Thr 402 Cys Cys Cys Cys Cys Cys Cys Cys Mainchain N compensates for pyrophosphate negative charge Glu 430 Thr Thr Thr Thr Thr Thr Thr Thr w a £ % ■ £ > ‘ J > “ w \ mm 1 1 Mainchain NH H-bonds w/ carbonyl at C2 of pyrimidine Val 440 Met Met Met Met Met Met Met Met Is ) 00 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 6. Common substrate binding amino acids in MAO and PAO. The function of the sixteen amino acids associated with the substrate binding pocket in PAO and their corresponding ones in MAO A and B across various MAO types is shown. Trout MAO (T), human (h-A), rat (r-A), mouse (m-A), and bovine (b-A) MAO A, human (h-B), rat (r-B), and mouse (m-B) MAO B, Identical amino acids are represented by an equal sign“- \ Amino acids selected for mutagenesis are highlighted. PAO T h-A r-A m-A b-A h-B r-B m-B Function in polyamine oxidase AA pos. Located at the tunnel bend. Sidechain oxygen close to inhibitor Glu 62 Gly Gly Gly Gly Gly Gly Gly Gly Funnel 'carboxylate ring' at entrance of active site tunnel Asp 117 Arg Arg Arg Arg Arg Arg Arg Arg :^c< M ltk> kylat0' H n a s i t e tunnel tP - •Glu 120 ?Asp Asp Asp A sp : :Asp. > a sp /Asp..1 '; Funnel ’ carboxylate ring' at entrance of active site tunnel Glu 1 2 1 = Asn = Asp Asn Asp = = Funnel 'carboxylate ring' at entrance of active site tunnel Glu 124 Ser Lys Lys Lys Lys Arg Gin Gin Mainchain carboxyl interacts with substrate Tyr 169 Thr = = = Ser Thr Thr Thr Located at the tunnel bend. Sidechain oxygen close to inhibitor Glu 170 Leu Leu Leu Leu Leu Leu Leu Leu Mainchain oxygen dose to inhibitor carbons Phe 171 = = = = = = = = Funnel 'carboxylate ring' at entrance of active site tunnel Asp 194 Thr Thr Thr Thr Thr Thr Thr Thr Funnel 'carboxylate ring' at entrance of active site tunnel Asp 195 M Thr A Ser Thr Thr Thr Thr Sidechain OH H-bonds with inhibitor secondary amine Tyr 298 Val Val Val Val Val Val Val Val *PI» » m m w m « » m msklYftwr Mainchain oxygen dose to inhibitor carbons Ser 404 Thr Thr Thr Thr Thr Thr Thr Thr Sidechain oxygen dose to inhibitor carbons Asn 405 Ala Ala Ala Ala Ala Ala Ala Thr Mainchain oxygen dose to inhibitor carbons Gly 438 = = = = = = = = ?:! 43? ‘■•"sW m t M mm 29 Figure 7. The FAD binding site. The conserved residues in MAO are marked by arrows. The interaction of Lys-300 with the N5 of FAD in PAO was studied in MAO by making the mutants A-K305A and B-K296A. The interaction of Trp-393 with dimethylbenzene moiety in PAO was studied by making mutants A-W397A and B- W388A. (Adapted from Binda et al., 1998). Trp-393 Lys-300 IGIy-57 Asn_59 \ l n h 2 v NH <N |s jH— O— C H N ^ T rp -6 0 OH Thr-402 H2g h2 o ------- .NH s ' / Glu-430 HN ^V al-400 Tyr-439 Ser-15 C = 0 / I Arg-43 NH — O — P = 0 / o nh2 nh2 -0 - 7 P = 0 ^ -— Y Arg-43 o ' : : N H h2 o h2 o OH OH OH NH Glu-35 Tyr-399 NH2. o = c Val-237 NH Ala-36 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 8. The substrate binding site. The three amino acids conserved with MAO within the U-shaped substrate binding site of PAO are market by arrows. Glu-120 sits at the entrance of the binding site and was mutated to alanine in MAO A and B C A DI 32 A, B-D123A). The aromatic sandwich of Phe-403 and Tyr-439 was studied by making mutants A-Y407F/S, A-Y444F/S, B-Y398F/S, and B-Y435F/S. (Adapted from Bindaetal., 1998). 3n h Phe-403 Gly-438 Glu-170 11 12 OH O ) Glu-62 Glu-170 Tyr-298 Ser-404^= O .... h 2n a .. Y Asn-405 Tyr-169 Tyr-439 Phe-171 Glur124 Asp-194 Glu-121 Asp-195 Glu-120 t 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS MAO A mutants We have assayed the seven MAO A mutants and the wild-type for activity using the MAO A substrate serotonin (5-HT) (Table 2). A-K305A and A-W397A did not show detectable activity even when the amount of protein in the assay tube was increased to about 100 times that of MAO A wild-type. These results indicate that Lys-305 and Trp- 397 in MAO A play a critical role in enzyme activity. A-Y407S exhibited no activity whereas A-Y407F had approximately 50% of wild- type activity. The Km value of A-Y407F was slightly increased. Similarly, mutant A- Y444S did not show any catalytic activity whereas the mutant A-Y407F had low activity. These results indicate that the two tyrosines at positions 407 and 444 can be replaced by phenylalanine to retain some activity but not by serine. This implies an important role for the phenyl group of tyrosine. A-D132A had an activity similar to that of the wild type and the Km value was slightly increased. This suggests that Asp-132 is not important for the catalytic activity of MAO A. We have also determined the inhibitor sensitivities of all the active mutants towards the MAO A specific inhibitor clorgyline and the MAO B specific inhibitor deprenyl (Table 3). For clorgyline, A-D132A and A-Y407F had the same sensitivity as the wild- type and A-Y444F showed about a 10-fold decrease in sensitivity. For deprenyl, A- D132A showed a slight decrease in sensitivity and A-Y407F and A-Y444F showed about a 10-fold decrease. Therefore A-Y444F shows a decreased sensitivity for both 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. inhibitors while A-D132A and A-Y407F shows a decreased sensitivity towards deprenyl only. This suggests that although these amino acids are not essential for enzyme activity, they do have an effect on the substrate binding site. Table 2. Activities o f MAO A active site mutants. The activity and affinity of MAO A wild-type and its mutants towards 5-HT is shown. Data are from at least two experiments ± SEM.*Activity was too low to determine Km A c t i v i t y K m nzym e (nm ol 5 -H T /m in /m g ) (pM 5-HT) 61.3 ± 3 . 3 86.3 ± 7.3 MAO A w i l d t y p e MAO A -D 132A MAO A-K3 05A MAO A-W3 97A MAO A -Y 4 0 7 F MAO A -Y 4 0 7 S MAO A -Y 4 4 4 F MAO A -Y 4 4 4 S 57.2 ± 2 . 5 N o t D e t e c t a b l e N o t D e t e c t a b l e 3 4 . 9 ± 1 . 9 N o t D e t e c t a b l e < 0.1 N o t D e t e c t a b l e 1 8 6 .6 ± 23 .6 N o t D e t e c t a b l e N o t D e t e c t a b l e 120 .0 ± 1 7. 6 N o t D e t e c t a b l e N o t D e t e r m i n e d * N o t D e t e c t a b l e 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3. Inhibition constants o f MAO A and B active site mutants. Clorgyline and deprenyl IC50 values for MAO A and B wild type and for the active mutants are shown. Data are from at least two experiments ± SEM C l o r g y l i n e IC50 D e p r e n y l IC 50 Enzym e (M) (M) MAO A w i l d t y p e 1 . 2 + 0 . 5 X 1 0 ' 9 2 . 3 + 1 . 2 X 1 0 ' 6 MAO A -D 132A 2 . 2 + 0 . 8 X 1 0 ' 9 8 . 9 + 4 . 2 X 1 0 ' 6 MAO A -Y 4 0 7 F 2 . 3 + 1 . 3 X 1 0 ' 9 2 . 5 1 . 5 X 1 0 ' 5 MAO A -Y 4 4 4 F 1 . 9 + 1 . 5 X 10~8 2 . 4 + 0 . 4 X 1 0 ' 5 MAO B w i l d t y p e 4 . 2 + 2 .1 X 10~7 3 . 3 + 1 . 4 X 1 0 ' 9 MAO B -D 123A 1 . 4 + 0 . 3 X 1 0 ' 6 1 . 1 + 0 . 4 X H O 1 CD MAO B -Y 3 9 8 F 3 . 3 ± 0 . 9 X 1 0 ' 6 5 . 1 + 2 . 0 X 1 0 ' 8 MAO B -Y 4 3 5 F 3 . 6 ± 1 . 0 X 1 0 ' 6 4 . 3 + 2 . 1 X 10*7 MAO B mutants The seven corresponding mutants were also made on MAO B and the activity towards the MAO B substrate PEA was assayed (Table 4). Interestingly, the results were similar to the MAO A mutants. B-K296A and B-W388A resulted in a complete loss of activity. B-Y398S was also inactive but B-Y398F retained activity with a slight increase in Km value. Similarly, B-Y435S was inactive and B-Y435F retained activity with an increase in Km . As for its MAO A counterpart, B-D123A had an activity similar to that of the wild type which suggests that Asp-123 is not important for the catalytic activity of MAO B. 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Al! three active mutants, B-D123A, B-Y398F, and B-Y435F showed a decrease in sensitivity towards clorgyline and deprenyl. B-Y435F showed an especially marked decrease in sensitivity of about 100 fold towards deprenyl. This suggests that these mutants affect the active site. Our results show that Lys-305, Trp-397, Tyr-407, and Tyr-444 in MAO A and their corresponding amino acids in MAO B, Lys-296, Trp-388, Tyr-398, and Tyr-435, play an important role in MAO activity. Based on the polyamine oxidase structure, Lys-305 and Trp-397 in MAO A and their corresponding amino acids in MAO B, Lys-296 and Trp-388 may be involved in the non-covalent binding of FAD to MAO. Tyr-407 and Tyr-444 in MAO A (Tyr-398 and Tyr-435 in MAO B) form an aromatic sandwich within the substrate binding site. Moreover the high similarity observed between the MAO A and MAO B mutants suggests that these amino acids have the same function in both isoenzymes and that the overall structure of MAO A and B and their active sites are similar. 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4. Activities o f MAO B active site mutants. The activity and affinity of MAO B wild-type and mutants towards PEAis shown. Data are from at least two experiments ± SEM Enzyme A c t . (nmol PEA/min/mg) K ,» (|OM PEA) MAO B w i l d t y p e 2 7 . 2 ± 3 . 0 2 .2 ± 0 . 5 MAO B-D123A 2 4 . 0 ± 2 . 2 5 .2 ± 2 . 1 MAO B-K296A N o t D e t e c t a b l e N o t D e t e c t a b l e MAO B-W388A N o t D e t e c t a b l e N o t D e t e c t a b l e MAO B-Y398F 1 9 . 7 ± 3 . 8 7 .8 ± 2 . 5 MAO B-Y398S N o t D e t e c t a b l e N ot D e t e c t a b l e MAO B-Y435F 3 . 5 ± 0 . 4 2 1 . 1 ± 2 . 0 MAO B-Y435S N o t D e t e c t a b l e N o t D e t e c t a b l e DISCUSSION The structure of the recently crystallized polyamine oxidase (PAO) was used as a model to study the residues involved in FAD attachment and the substrate binding site of MAO. PAO shares a 20% amino acid identity with MAO and we have compared amino acids that are conserved between PAO and all the cloned MAOs: human, rat, mouse, and bovine MAO A, human, rat, and mouse MAO B, and trout MAO. According to the PAO structure, there are fifteen amino acids associated with FAD binding (Fig. 5) and sixteen amino acids associated with the substrate binding pocket (Fig. 6 ). Of the fifteen amino acids associated with FAD in PAO, six (Asn-59, Trp-60, Tyr-399, Thr-402, Glu-430, and Val-440) are nonconserved with MAOs, and five (Ser- 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15, Glu-35, Ala-36, Arg-43, and Gly-57 in PAO) are conserved with MAOs but are within the general FAD signature motif of flavoproteins near the N-terminus. Val-237 and Tyr-439 of PAO are conserved with all MAO but exert their function via their mainchain atoms (Binda et al., 1999) (Fig. 7). Mutagenesis on these residues would therefore not be useful in ascertaining their function. Thus there are only two amino acids in PAO which are conserved among all MAOs and bind to FAD through their sidechains: In PAO, Lys-300 makes an indirect bond with the isoalloxazine moiety of FAD via a water molecule and Trp-393 binds the dimethylbenzene portion of FAD. FAD binding site Mutating the corresponding amino acid of Lys-300 in PAO to alanine in MAO A and B to produce A-K305A and B-K296A respectively results in the complete inactivation of the enzymes. Therefore this conserved lysine is necessary for enzyme activity and may therefore have the same interaction with the FAD of MAO A and B as it does in PAO. Mutating the equivalents of Trp-393 in PAO to alanine in MAO A and B also produces inactive enzymes. Trp-393 in PAO has extensive Van der Waals interactions with the dimethylbenzene portion of FAD and its equivalent in MAO A and B may have the same function. Taken together, these results suggest that Lys-305 and Trp-397 in MAO A and their corresponding residues in MAO B, Lys-296 and Trp-388, are essential for the activity of the enzyme. They may play an important role in the non-covalent binding or in the 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. incorporation of FAD which is disrupted when they are mutated to alanine. FAD attachment to MAO has been shown to be disrupted when the amino acids for covalent attachment, Cys-406 in MAO A and 397 in MAO B, are mutated to serine (Chen et al., 1994). It has also been shown that FAD does not get incorporated when noncovalently binding amino acids are mutated (Kirskey et al., 1998, Zhou et al., 1998). This implies that FAD incorporation to MAO requires both covalent and non-covalent interactions with the enzyme. Substrate binding site Of the sixteen amino acids associated with the PAO substrate binding pocket, only 5 (Glu-120, Phe-171, Phe-403, Gly-438, and Tyr-439) are conserved with MAO A and B from different species (Fig. 6 ). Glu-120 is located at the entrance o f the substrate binding site of PAO which is a U- shaped tunnel but does not appear to have an interaction with either the FAD or the substrate (Fig. 8 ). The corresponding amino acid are Asp-132 in MAO A and Asp-123 in MAO B. Mutating them to alanine had a negligible effect on enzyme activity indicating that this residue is not essential for enzyme function. Phe-403 and Tyr-439 are the only two amino acids in the substrate binding site of PAO that are conserved with MAO. In PAO they form an ‘aromatic sandwich’ which consists of these two residues flanking the opposite sides of the substrate binding site in a parallel fashion. Furthermore, the hydroxyl group o f Tyr-439 interacts directly with 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the inhibitor (Fig. 8 ). When their corresponding residues in MAO A, Tyr-407 and Tyr- 444, and MAO B, Tyr-398 and Tyr-435, are mutated to serine the enzyme loses all activity, however when they are mutated to phenylalanine the enzyme remain active. This indicates that indeed it is the aromatic portion and not the hydroxyl portion that is essential for activity and suggests a similar aromatic sandwich structure in MAO. Interestingly, A-Y444F and B-Y435F have very low activity compared to the wild- type while the A-Y407F and B-Y398F mutants had similar activity to the wild-type. This indicates a more important function that the hydroxyl groups of Tyr-444 and Tyr- 435 than those of Tyr-407 and Tyr-398 for substrate binding. This is in agreement with the structure of the substrate binding site of PAO in which the hydroxyl group of Tyr- 439, the PAO equivalent of Tyr-444 and Tyr-435 in MAO A and B, interacts directly with the bound inhibitor (Fig. 8 ). The mutants A-D132A and B-D123A resulted in only a slight decrease in substrate affinity. Therefore Asp-132 and Asp-123 in MAO A and B are not important for enzyme activity. Taken together, these results show that with the exception of two mutants (A- D132A and B-D123A) amino acids conserved between PAO and MAO and whose function is mediated by the amino acid side chain result in inactive MAOs when mutated to nonconserved residues. This suggests that MAO and PAO have the same overall structure, and at least one common feature within the substrate binding site. It is also noteworthy that the all mutations produced a very similar effect in MAO A and 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MAO B. This suggests that FAD binding and the substrate binding site in the two MAO isoforms are highly similar. 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2) Phe-208 and Ile-199 in human monoamine oxidase A and B do not determine substrate and inhibitor specificities as in rat INTRODUCTION AND RATIONAL In order to determine the region(s) responsible for the distinct substrate and inhibitor specificities of MAO A and B we and other groups have made point mutants and chimeric MAO constructs exchanging corresponding portions of the isoenzymes (Cesura et al., 1996; Chen et al., 1996; Gottowik et al., 1993, 1995; Tsugeno et al., 1995; and Wu et al., 1993). It has been shown in our lab that amino acid segments 161- 375 in human MAO A and 152-366 in human MAO B contain part of the domain responsible for determining specificity (Grimsby et al., 1996). It was later discovered that reciprocally switching amino acids Phe-208 in rat MAO A and its corresponding residue in rat MAO B, Ile-199, was sufficient to switch the substrate and inhibitor specificities of rat MAO A and B (Tsugeno and Ito, 1997). To determine if the human MAO equivalents of these two residues, which are located within the segments in our earlier chimerics MAO AB161- 375A and MAO BA[52- 366B (Grimsby et al., 1996), are responsible for the change in substrate and inhibitor specificities we have observed in the human MAO chimerics, we have made the equivalent point mutations in human MAO A and B. We have also made additional chimerics by reciprocally switching segments 159-214 and 150-205 in human MAO A and B respectively (Fig. 9). These chimerics represent the N-terminal portion of the switched internal segments of our previous chimerics and include the amino acids Phe-208 in MAO A and 1199 in MAO B. This will allow us to determine if this amino acid pair has the same specificity 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. determining function in rat and human MAO, and if not, whether substrate and inhibitor preference were determined by the amino acids near these two residues. Figure 9. Construction o f MAO A and B point mutant and chimeric enzymes. The mutations were performed on full length human MAO A and B cDNA subcloned into the pYES2 yeast expression vector as detailed in "experimental procedures". MAO A and MAO B are represented by an open box and a filled box respectively. Restriction endonuclease sites introduced by mutagenesis are marked by asterisks. Locations of restriction sites are shown as the amino acid number underneath. Locations of the point mutations MAO A-F208I and MAO B-I199F are indicated by a "t". icI 214 iV lA O A V - Introduce unique BglH site Introduce unique MscI site Introduce unique BglH site -H 159 214 7 Phe-208 ]----- 1 T Ile-199 Double digest with Bgin and MscI and cross-ligate small and large fragments - i H 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS Our results show that only MAO A and MAO A-F208I oxidize 5-HT (Table 5). MAO B and the remaining mutants do not. This is consistent with previous work (Grimsby et al., 1996; Tsugeno and Ito, 1997) in which 5-HT oxidation was not detectable when the expression level o f MAO B was not abundant. However, both MAO A and B as well as all mutants and chimerics exhibited significant activities when PEA was used as a substrate. This result indicates that all constructs were adequately expressed. Table 5. Activities o f chimeric MAOs and mutants A-F208I, B-I199F. Catalytic activities for the oxidation of 5-HT and PEA by wild type, point mutant, and chimeric MAOs is shown. 1 to 15 pg of mitochondria from yeast expressing MAO were incubated with saturating concentrations of labeled 5-HT or PEA in a 1 ml total volume of 50 mM sodium phosphate buffer pH 7.4 for 20 min at 37°C. Amount of oxidized product was measured by scintillation counter. Results are Mean ± S.E.M. of three experiments. N/D: activity not detectable up to 2.5 mM substrate Activity (nmol/min/mg protein) Enzyme 5-HT oxidation PEA oxidation MAO A 2 5 .2 ± 0 .6 4 6 . 8 6 ± 0 .3 9 MAO B N/D 0 .53 + 0 .06 MAO A-F208I 1 3 .1 ± 1 .4 3 .52 + 0 .50 MAO B-I199F N/D 3 .3 1 + 0 .05 MAO AB159.214A N/D 1 .5 4 + 0 . 2 2 MAO BA150-205B N/D 0 .2 8 + 0 .03 Kinetic constants which are independent of enzyme concentration, Km and IC50, were used to compare enzyme affinities for substrates and inhibitors respectively. Since 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MAO A and B have a preference for clorgyline and deprenyl respectively we have used the clorgyline/deprenyl IC50 ratio to demonstrate the inhibitor preference. The wild type form of MAO A oxidizes both 5-HT and PEA with Km values of 57.1 ± 4.8 pM and 67.2 ± 6.7 pM respectively (Table 6 ). Point mutant MAO A-F208I, like MAO A, oxidizes both substrates. However its 4-fold higher Km values of 219.3 ± 28.1 and 258.0 ± 10 pM for 5-HT and PEA respectively suggest that this point mutation decreased the affinity of the enzyme to both substrates equally. Table 6 . Kln values o f chimeric MAOs and mutants A-F208I, B-Il 99F. The Km values for MAO A and B, the point mutants MAO A-F208I and MAO B-I199F, and the chimerics AB159.214A and BA150- 205B were determined on the mitochondrial fraction of yeast homogenates as detailed in "experimental procedures". Results are Mean ± S.E.M. of three experiments. N/D: Activity not detectable up to 2.5 mM substrate. K m ( p M ) Enzyme 5-HT PEA MAO A 5 7 . 1 ± 4 . 8 6 7 . 2 + 6 . 7 MAO B N/D 3 . 0 + 0 . 3 MAO A-F208I 2 1 9 . 3 ± 2 8 . 1 258 . 0 ± 10 MAO B-I199F N/D 5 . 9 ± 0 . 6 MAO ABi59-2[4A N/D 6 4 6 . 0 ± 136 MAO BA150- 205B N/D 5 . 3 ± 0 . 7 The clorgyline and deprenyl IC50 values for MAO A were 5.1 ± 0.7 x 10'9 M and 1.0 ± 0.2 x I O '6 M respectively (Table 7), resulting in a clorgyline/deprenyl IC50 ratio of 5 x 10'3. For MAO A-F208I, the clorgyline and deprenyl IC50 values were 3.5 ± 1.5 x 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I O '9 and 1.3 ± 0.7 x 10° M respectively (Table 7), indicating no change in affinity for the MAO A inhibitor clorgyline and a 10 fold decrease in affinity for deprenyl compared to the wild type, and resulting in a clorgyline/deprenyl IC50 ratio of 3 x 1C T 4 (Table 7). This ratio is clearly closer to that of MAO A than MAO B. Overall, the pharmacological profile of MAO A-F208I is clearly similar to that of MAO A and did not exhibit a substrate or inhibitor preference shift towards MAO B. Table 7. ICso values o f chimeric MAOs and mutants A-F208I, B-I199F. The IC50 inhibition constants for clorgyline and deprenyl of wild type, point mutant, and chimeric MAOs. IC50 values were determined as detailed in "experimental procedures". PEA was used as a substrate. The concentration of inhibitor required for 50% inhibition of MAO activity was calculated by Hill analysis. Results are mean of 3 experiments ± S.E.M. ICso (M ) Enzyme Clorgyline L-Deprenyl Clo./Dep. ratio MAO A 5 . 1 + 0 . 7 X 1 0 ' 9 1 . 0 + 0 . 2 X 10*6 5 x 10 MAO B 6 . 8 + 0 . 5 X 10~6 2 . 7 + 0 . 5 X 10*8 252 MAO A-F208I 3 . 5 + 1 . 5 X 1 0 ' 9 1 . 3 + 0 . 7 X 1 0 ' 5 3 x 10 MAO B-I199F 3 . 8 1 . 3 X 1 0 ' s 2 . 1 ± 0 . 5 X 1 0 ‘9 1810 MAO A B159-214A 3 . 5 + 0 . 2 X 1 0 ' 8 2 . 4 ± 1 . 1 X 10~s 2 x 10 MAO BA159-205B 3 . 1 1 . 9 X 10*5 2 . 0 ± 0 . 1 X 10~7 155 MAO B oxidizes PEA with a Km o f 3.0 ± 0.3 jiM and the oxidation of 5-HT was not detectable (Table 6 ). The 5-HT and PEA oxidation parameters o f point mutant 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MAO B-I199F were similar to those of MAO B. It could not oxidize 5-HT and the Km for PEA was 5.9 ± 0.6 pM, which is similar to that of MAO B. MAO B was inhibited by clorgyline and deprenyl with ICso values of 6 . 8 ± 0.5 x 10' 6 M and 2.7 ± 0.5 x 1 0 ’8 M respectively, resulting in a clorgyline/deprenyl IC50 ratio of 252 (Table 7). The MAO B-I199F ICso parameters for clorgyline and deprenyl were 3.8 ± 1.3 xlO'6 M and 2.1 ± 0.5 x 10'9 respectively, showing a 10-fold increase in deprenyl affinity compared to the wild type and no change in affinity for clorgyline. The resulting clorgyline/deprenyl ICso ratio of 1810 is closer to that of MAO B than MAO A. Therefore the mutation did not change enzyme affinity for 5-HT, PEA and clorgyline, and increased affinity for deprenyl. Overall, MAO B-I199F kinetic properties were similar to the parent enzyme and did not acquire MAO A-like properties. Unlike MAO A-F208I, the chimeric AB159.214A enzyme was incapable of oxidizing 5-HT. The Km for PEA of 646 ± 136 pM (Table 6 ) was 10 fold higher than MAO A. Although losing catalytic activity towards 5-HT makes it similar to MAO B, the fact that the Km value for the MAO B preferred substrate PEA was increased by about 10 fold indicates a decrease in affinity for both 5-HT and PEA and not a shift in specificity. The clorgyline/deprenyl IC50 ratio of 2 x 1 0 * 2 (calculated from clorgyline and deprenyl IC50 values of 3.5 ± 0.2 x 10'8 M and 2.4 ±1.1 x 10’6 M respectively) is similar to that of MAO A. Therefore replacing region 159-214 in MAO A with the corresponding region in MAO B decreased the enzyme affinity for 5-HT, PEA and clorgyline but did not confer MAO B-like properties to the chimeric construct. 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Similarly, replacing amino acid segment 150-205 of MAO B with the corresponding region of MAO A to produce chimeric BA150- 205B did not confer 5-HT oxidizing ability and the PEA Km value of 5.3 ± 0.7 p.M was similar to that of MAO B. The ICso values for both clorgyline and deprenyl increased by about 10 fold compared to MAO B, 3.1 ± 1.9 x 10'5 M for clorgyline and 2.0 ±0.1 x 10'7 M for deprenyl, resulting in a IC50 ratio of 155, similar to that of MAO B. Therefore MAO BA150- 205B had a decreased affinity for both inhibitors, did not exhibit a B to A preference switch and generally had similar properties to MAO B. Taken together, our results indicate that reciprocally switching amino acids Phe-208 and Ile-199 in MAO A and B respectively does not switch substrate and inhibitor specificities of the enzymes, nor does reciprocally switching the amino acid segments 159-214 in MAO A and 150-205 in MAO B, which include Phe-208 and Ile-199. The point mutations as well as the chimerics did have differences in affinities for some substrates and inhibitors indicating that the changed amino acids may have some effect on the conformation of the enzyme. Thus substrate and inhibitor preferences in human MAO A and B may not be governed by this amino acid as in the rat (Tsugeno and Ito, 1997). DISCUSSION We have previously reported that the substrate and inhibitor preferences of human MAO A and B may be conferred by a middle portion stretching from amino acids 161 to 375 in MAO A and its corresponding segment ranging from amino acids 152 to 366 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in MAO B (Grimsby et al., 1996). Reciprocally switching these two segments results in one chimeric, AB161- 375A (MAO A with segment 161-375 replaced by the corresponding MAO B segment), acquiring B-like pharmacological properties and an inactive chimeric, BA152- 366B. This switched segment included the human equivalents of amino acids Phe-208 and Ile-199 of rat MAO A and B respectively, which were shown responsible for substrate and inhibitor preferences of the two isoenzymes (Tsugeno and Ito, 1997). To investigate if the changed preference we have observed in chimeric AB161- 375A was the result of switching these two amino acids we have made mutants MAO A- F208I and MAO B-I199F on human MAO’s. These two mutants exhibited similar kinetic properties to the parent enzymes and despite slight changes in the kinetic parameters, the IC50 ratio of clorgyline/deprenyl remained close to the parent enzymes. This indicates that interchanging Phe-208 and Ile-199 of human MAO A and B respectively does not switch their substrate and inhibitor preferences. These results stand in contrast to the effects these mutations had on rat MAO. Rat MAO A-F208I had lost its ability to oxidize 5-FIT, its Km for PEA was decreased by 50% and its affinity for clorgyline and deprenyl were the reverse of wild type rat MAO A and similar to rat MAO B. In fact, it acquired a MAO B-like pharmacological profile. Activity for 5-HT oxidation became detectable in rat MAO B-I199F which also exhibited an increased Km for PEA becoming more MAO A-like. These results suggest that amino acid Phe-208 and Ile-199 in rat MAO A and B respectively have a different function than the corresponding amino acids in human MAO. 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, the kinetic properties of MAO A and MAO A-F208I suggest that the F208I mutation has had a effect on the active site and decreased its affinity for 5-HT, PEA, and deprenyl. While the I199F mutation in MAO B appears to have had a lesser effect. Although the switched residues are both hydrophobic, phenylalanine is much bulkier than isoleucine. For this reason the I199F mutation was expected to be more disruptive to the structure of the active site than the F208I mutation and would thus have a greater effect on the kinetics constants. The observation that the opposite was the case suggests that the aromatic ring plays an important role in the active site of human MAO but not a preference determining role as demonstrated in rat. Rat and human MAO A have a 88% sequence identity and a 92% sequence homology; rat and human MAO B have a 88% sequence identity and a 93% sequence homology. It is unlikely that a MAO isoenzyme has a significantly different active site between rat and human. However rat and human MAO have an opposite pharmacology for dopamine (Glover and Sandler, 1977) indicating their active sites may not be identical. We compared the amino acids which may be important for the conformation of the enzyme between human and rat MAO's: Cysteine, which may form disulfide bridges, and Proline, which induces a turn. All cysteines in human MAO A and B are conserved in their rat counterpart, however rat MAO's have additional cysteines and non conserved prolines. Ala-289 in human MAO A is Proline at the corresponding position in rat MAO A, Gly-515 and Arg-526 are changed to Cysteine. Comparing human and rat MAO B, Ser-26 and Arg-38 in human are changed to Cysteine in rat, Pro-98 is 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. changed to Alanine, Tyr-337 is changed to Cysteine, and Ser-465 is changed to Proline in rat. This indicates that despite the high sequence identity there may be differences in the secondary or tertiary structure, which could explain the different substrate and inhibitor specificity of mutants derived from rat and human MAO isoenzymes. To investigate if the regions neighboring Phe-208 and Ile-199 are responsible for substrate and inhibitor preference in human MAO A and B we have constructed chimeric enzymes by reciprocally switching segments 159-214 and 150-205 in human MAO A and B respectively. Chimerics AB159-214A and BA150-205B differ by 15 amino acids from their parent enzymes (including Phe-208 and Ile-199). MAO AB159.214A had slightly different kinetic parameters than MAO A and MAO BA150-205B was similar to MAO B, with no change in substrate or inhibitor preference except for deprenyl. The human chimeric AB161- 375A was constructed by switching segment 161-375 of human MAO A by its corresponding region in MAO B (Grimsby et al., 1996). It acquired MAO B-like properties since this chimeric exhibited a lower Km for PEA and the clorgyline/deprenyl IC50 ratio became like that of MAO B. This result shows that MAO AB159.2t.jA retained MAO A-like properties which suggests that the B-like properties exhibited by AB161- 375A may be due to segment 215-375. The difference between the inactive chimeric BA 152- 366B, and the active chimeric BAt5o - 205B is amino acid segment 206-366. This may indicate a necessary interaction of this region with the active site of MAO B in order for the enzyme to be active. In summary, this study shows that Phe-208 and Ile-199, which are responsible for substrate and inhibitor preference in rat MAO A and B, are not responsible for 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. preferences in MAO A and B. And in human MAO the preference determining region may be within the corresponding segments 215-375 in MAO A and 206-366 in MAO B. Reciprocally interchanging amino acids that are conserved between species and different between MAO subtypes within these segments may reveal the residues important for preference in human MAO. In the next study we explore this region for specificity determining residues. 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3) Substrate and inhibitor specificities of human monoamine oxidase A and B can be switched by a single amino acid INTRODUCTION AND RATIONAL In the previous section we have found that the residues which may be important for specificity in human MAO are within a 161 amino acid middle segment (amino acids 215-375 in MAO A and 206-266 in MAO B). This segment contains 32 amino acids that are non-conserved between human MAO A and B of which 15 are conserved among the different species of MAO A or B. Trout MAO shares a 70% and 71% amino acid identity with MAO A and B respectively. However since the substrate and inhibition profile of trout MAO is much closer to that of MAO A than MAO B (Chen et al., 1994), classifying it as such allows us to reduce the number of amino acids potentially responsible for specificity from 15 to 5 (Fig. 10). Of these, Glu-286 in MAO A and Leu-361 in MAO B, have previously been mutated to the corresponding residue in the other MAO without exhibiting differences in specificity from the parent enzyme for the substrates 5-HT and PEA and the inhibitors clorgyline and deprenyl (unpublished data) suggesting that the residues at these positions may not determine specificity. For the other 3 amino acids we have made 6 reciprocal mutants. We have found that when two corresponding amino acids, Ile-335 and Tyr-326 in human MAO A and B respectively, were reciprocally interchanged, the substrate and inhibitor specificities were also switched. This result suggest that Ile-335 and Tyr-326 in human MAO A and B respectively play a key role in conferring substrate and inhibitor specificities in human MAO. 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 10. Multiple sequence alignment o f the putative specificity determining 166 amino acid segment. The putative 161 amino acid segment responsible for specificity in human MAO A and B (residues 215-375 in MAO A and 206-366 in MAO B) is aligned from mouse, rat, and bovine MAO A, mouse and rat MAO B, and trout MAO. The 15 amino acids that are non-conserved between MAO A and B subtypes and concurrently conserved among all the different species within a subtype are marked by asterisks. When trout MAO is classified as a “MAO A subtype” because of similar kinetics (Chen et al., 1994), the remaining 5 amino acids non-conserved between subtypes and conserved among the different species of a subtype are shaded. The 3 corresponding amino acid pairs selected for reciprocal interchange by mutagenesis are indicated above the boxed amino acids. MAO A-T24S MAO B-X236 Mouse MAO A 215 QERKFVGGSGQISEQIMVLLGDKVKLSSPVTYIDQTDDNIIIETLNHEHYECKYVISA 272 Rat MAO A 215 V:::::G: : : : * » » * :::::::V:: : : : : 272 Bovine MAO A 215 V::R::Q: : :R : :R: :hv :SSE::TV::::R L ::R:::: 272 Human MAO A 215 V::R::D: ::Q : :NH : iHV :SS: :::::::: : : :: : : : :N 272 Trout MAO 208 : L: :S ::tCMAKE :ER :ME: :YK: ::G:MVEV::::K T KA: : : :V 265 Mouse MAO B 206 : I: V::R:KDI : :R : : ER :IH: ::GE: V:VK:::: I :A:: : : : 263 P.at MAO 3 206 :I: V::R:KDI : : R : :ER : XH: : :GE:VWK: : : : I :A:::: : 263 Human MAO B 206 : : : V::R:MD: : :R : :ER : X: : ::RE: VLV: : : : : M : A: : : : : 263 MAO A-D328 MAO B-Q319 Mouse MAO A 273 IPPVLTAKIHFKPELPPERNQLIQRLPMGAVIKCMVYYKEAFWKKKDYCGCMIIEDEE 330 Rat MAO A 273 : : I : : : . s j :::::::: f . S 330 Bovine MAO A 273 : :T tR.jj S:::::: : : :::::M:: : . £ 330 Human MAO A 273 : :T : R : . s ! A: : : : ::: : :::::M:: : D 330 Trout MAO 266 T: G NL:M :N: s :L:::::H V S : : : :I:::R N: R: :G: : :T V i : 323 Mouse MAO B 264 : :A GM: :YSAB ML:::::S V L S::::::: :R P: R:::F::T V G : 321 Rat MAO B 264 : : :GM: :HS:P IL:::::T V L S: ::::::: : P: R:::F::T V G : 321 Human MAO B 264 : :T GM: : :N:I> MM:::M:T V L S: : : : X :: : : P: R:: : : : :T : DG : 321 • • • • • • * • MAO A-13 3 5 MAO B-Y326 Mouse MAO A 331 APISITLDDTKPDGSMPAIMGFILARKAERLAKLHKDIRKRKICE 375 Rat MAO A 331 Ar ::L:::::::::: :D: : 375 Bovine MAO A 331 : £ ::L:::::::::: :D::::V:: : : : 375 Human MAO A 331 : : ::L:::::::::: :D:::::: :E: : . eK: : : 375 Trout MAO 324 GL :TV: ::::::::: CRK:CG:T:EE: t ' K R : : 368 Mouse MAO B 322 AX :TYA::::::::H :RK:VR:T:EE:E: :L: 366 Rat MAO 3 322 AY AGCA::::::::H :RK:VR:T:EE:E::L: 366 Human MAO B 322 :VAY ::::::E:NYA::::::::H :RK::R:T:EE:EK:L: 366 # * # * * 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS We have reciprocally interchanged amino acids Thr-245, Asp-328, and Ile-335 in human MAO A with their corresponding amino acids in human MAO B individually to produce the mutants A-T245I, A-D328G, and A-I335Y. Their equivalent mutants in MAO B (B-I236T, B-G319D, and B-Y326I) have also been made. Wild type MAO A and B as well as the six mutants were successfully overexpressed in insect cells using recombinant baculovirus. The turnover number (£cat) and the affinity (Km ) towards the MAO A specific substrate 5-HT and the MAO B specific substrate PEA were determined. We have used the specificity constant, £C a/Km , to depict the specificity of an enzyme towards 5-HT or PEA. As shown in Table 8, MAO A wild-type had a 6-fold higher kc a l for 5-HT than for PEA but a similar Km for both substrates resulting in a £C a/Km value for 5-HT that is about 7 times that of PEA (Fig. 11). MAO B wild-type, on the other hand, had a 19-fold higher A 'c a t for PEA than for 5-HT and a much lower Km for PEA resulting in a f c ca t/Km for PEA that is about 40,000 times that for 5-HT. Similarly, MAO A and B had a higher & c a t/Km for 5-HT and PEA respectively. These results are consistent with literature findings which classify 5-HT as MAO A-specific and PEA as MAO B- specific (Chen et al., 1996, Grimsby et al., 1996, and Gottowik et al., 1997). In contrast to MAO A, mutant MAO A-I335Y showed a higher & cat for PEA than for 5-HT. It also exhibited a 35-fold increase in its Km for 5-HT to 2801 pM (which is similar to the 5-HT Km of MAO B of 3891 pM) thus the Km for 5-HT was lower than 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for PEA (Table 8). This produced a larger A r c at/Km for PEA than for 5-HT (Fig. 11). In effect, A-I335Y acquired a MAO B-like substrate specificity. 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 8. kau and Km values o f A-1335Y and H-Y326I, The kr,„ and K,„ values were determined as described in “Experimental Procedures” for the MAO A preferring substrate serotonin (5-HT) and the MAO B preferring substrate p-phenylethylamine (PEA). The values given are the means of at least 3 experiments ± S.E.M. Enzyme 5 •kcat -HT (min'1) PEA 5 -HT K m (pM) PEA MAO A w i l d t y p e 6 7 .4 ± 3 . 4 1 1 .2 + 0 . 4 80 ± 4 91 ± 4 MAO A -I335Y 0 . 7 ± 0 . 1 1 . 8 ± 0 . 3 2801 ± 6 87 ± 15 MAO B w i l d t y p e 5 . 1 ± 0 . 1 9 8 .4 + 5 . 2 3891 ± 17 1 . 9 + 0 . 1 MAO B-Y326I 2 1 . 3 ± 3 .3 27.3 ± 3.5 569 + 32 9.5 ± 1.5 C \ Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 11. /:c a ,/Km values of A-I335Y and B-Y326I. The specificity constants of wild-type MAO A and B and mutants MAO A-I335Y and MAO B-Y326I for the substrates 5-HT and PEA has been calculated from the data in table 8. 5-HT is represented by the black bar. PEA is represented by the gray bar. 51.8 MAO A MAO A-I335Y MAO B MAOB-Y326I Mutant B-Y326I had similar A r c a t values for 5-HT and PEA and, compared to MAO B, exhibited a 7-fold decrease in Km for 5-HT and a 5-fold increase in Km for PEA (Table 8 ). The resulting A r c a t/Km (Fig. 11) indicate that this mutant retained MAO B-like substrate specificities. However, the £c a t/Km of the mutant was only about 75-fold higher for PEA than for 5-HT compared to about a 40,000-fold difference in the MAO B wild-type. Therefore, eventhough B-Y326I retained a higher specificity for PEA than for 5-HT it exhibited a significant shift in specificity towards MAO A. In summary, Ile- 335 in MAO A and Tyr-326 in MAO B play an important role in the substrate specificity of human MAO A and B. A switch in sensitivities for MAO A and B irreversible inhibitors clorgyline and (-)- deprenyl were also observed (Fig. 12). MAO A has an IC50 value of 1.2xl0'9 M for clorgyline and 1.3xl0‘6 M for deprenyl while MAO B has an IC50 value of 6.3xl0'7 M for clorgyline and 4.3xl0*9 M for deprenyl. Compared to MAO A, A-I335Y exhibited a large decrease in sensitivity towards clorgyline (ICso=7.lxlO'6 M) and a large increase in sensitivity towards deprenyl (IC5o=l.2 xl(T7 M) (Fig. 12). Thus the inhibitor sensitivity of this MAO A mutant became MAO-B like. Similarly, MAO B-Y326I was MAO A-like and was more sensitive to clorgyline (IC5o=2 .8 xlO*s M) than deprenyl (IC5o= 1.8x1 O '7 M) (Fig. 12). Therefore Ile-335 and Tyr-326 determine clorgyline and deprenyl sensitivities. We have also studied enzyme sensitivity towards the reversible inhibitors Ro 41- 1049 (MAO A specific) and Ro 16-6491 (MAO B specific). As shown in Figure 13 A, A-I335Y had a much lower sensitivity towards Ro 41-1049 than did MAO A 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (ICso=5.6xlO'8 M for MAO A, 2.5x1c4 M for A-I335Y), becoming MAO B-like. Similarly, B-Y326I exhibited a higher sensitivity for the MAO A specific inhibitor Ro 41-1049 than did MAO B (IC50=2.5xlC 4 M for MAO B, 2.5xl0'6 M for B-Y326I) and became more like MAO A. However for the MAO B specific inhibitor, Ro 16-6491, both mutants exhibited a decrease in sensitivity when compared to their parent enzyme and no reversal in specificity was observed. The other two pairs of mutants, A-T245I and B-I236T; and A-D328G and B- G319D, showed a decrease in A r ca t values for both 5-HT and PEA compared to their parent enzymes. A-T245I and A-D328G showed a £cat of 12.6 ±1.6 and 1.6 ± 0.2 min*1 respectively for 5-HT (compared to 67.4 ± 3.4 min*1 for MAO A wild-type) and 4.2 ± 0.5 and 0.3 ± 0.1 min*1 for PEA (compared to 11.2 ± 0.4 min-1 for MAO A wild-type). B-I236T and B-G319D showed kc a t values of 1.8 ± 0.1 and 2.2 ±0.1 min"1 respectively for 5-HT (compared to 5.1 ±0.1 for MAO B wild-type) and 19.4 ± 0.8, and 14.3 ± 2.0 min' 1 for PEA (compared to 98.4 ± 5.2 for MAO B wild-type). They however retained the Km values for both substrates, thus resulting in unaltered specificities. Their IC50 values for clorgyline, deprenyl, Ro 49-1049 and Ro 16-6491 were also unchanged compared to their parent enzyme. These results suggest that the mutations decrease the catalytic activity by either directly or indirectly affecting the active site. However they do not affect substrate or inhibitor specificity. It should be noted that even though the substrate and inhibitor specificities of A- I335Y and B-Y326I were switched, their kinetic and inhibitory constants were not 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. identical to those of the other MAO: The Km values for PEA were not greatly affected. The IC50 values for the inhibitors shift toward but do not become identical to the opposite MAO and no specificity change was observed for Ro 16-6491. This suggests that other amino acids also play a role in determining substrate and inhibitor specificities. In summary, the MAO A mutant A-I335Y acquired kinetic parameters similar to those of MAO B. Similarly, the MAO B mutant B-Y326I acquired kinetic parameters more like those of MAO A. Therefore our results suggest that Ile-335 in MAO A and its corresponding residue in MAO B, Tyr-326, play an important role in conferring substrate and inhibitor specificities to human MAO A and B. 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 12. Clorgyline and deprenyl inhibition curves o f A-I335Y and B-Y326I. The clorgyline (A) and deprenyl (B) inhibition curves of wild-type MAOs and the MAO mutants which exhibited a change in inhibitor sensitivity, A-I335Y and B-Y326I, are plotted as percent inhibition versus log inhibitor concentrations. Error bars represent the S.E.M. of 3 experiments. The symbols ■, □, •, o represent MAO A, A-I335Y, MAO B, and B-Y326I respectively. A log [clorgyline] (M) 1 0 0 -I 80 • 60 • 40 • 20 « •H •H • H -8 -7 -6 -5 -4 -3 -11 -10 log [deprenyl] (M) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 13. Ro 41-1049 and Ro 16-6491 inhibition curves o f A-I335Yand B-Y326I. The Ro 41-1049 (A) and 16-6491 (B) inhibition curves of wild-type MAOs and the MAO mutants, A-I335Y and B-Y326I, are plotted as percent inhibition versus log inhibitor concentrations. Error bars represent the S.E.M. of 3 experiments. The symbols ■, □, •, o represent MAO A, A-I335Y, MAO B, and B-Y326I respectively. 1 0 0 -I 80 • 60 - 40 - 20 - •rl •H -8 -7 -6 -4 -3 -2 -1 log [Ro 41-1049] (M) a 1 0 0 1 • h 80 • 4J 2 60 ■ 2 40 ■ C J •rl 20 • o 'P n . 9 -7 -5 -3 - 1 log [Ro 16-6491] (M) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DISCUSSION Using site-directed mutagenesis, we have constructed 6 MAO mutants by reciprocally interchanging 3 corresponding amino acid pairs in human MAO A and B within a region thought to be important for conferring substrate and inhibitor specificities. Corresponding mutant pair, A-I335Y and B-Y326I, exhibited the opposite specificities of their parent enzymes MAO A and B respectively. A-I335Y became more like MAO B while B-Y326I became more like MAO A. This suggests that Ile- 335 in MAO A and Tyr-326 in MAO B have an important function in determining the specificities of human MAO A and B. It is possible that in human MAO A the binding of the substrate or inhibitor may be facilitated by hydrophobic interactions with Ile-335 whereas in human MAO B, it may be mediated by aromatic stacking or hydrogen bonding with the hydroxyl group of Tyr-326. It is also possible that these two amino acids influence the 3-dimentional structure of the enzyme and affect substrate and inhibitor specificities. However this binding interaction may not be generalized to MAOs of other species. It was reported that switching Phe-208 and Ile-199 in rat MAO A and B respectively results in a partial inversion of specificities for some substrates and inhibitors (Tsugeno and Ito, 1997). It was suggested that the structural feature responsible for determining specificity in rat MAO A was the aromatic ring of Phe-208 and for rat MAO B it was the aliphatic side chain of Ile-199. This is the reverse of our present observation in human MAO in which the aliphatic residue is in MAO A (Ile-335) and the aromatic 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. residue is in MAO B (Tyr-326). Even though the data from the rat MAO mutants consisted of only Km values to ascertain substrate specificity and a single inhibitor concentration to determine inhibitor specificity; when compared to our results, it may point to a MAO species difference in the way specificity is determined. In fact, we have previously found that the same amino acid substitutions made on human MAO, A- F208I and B-I199F, did not result in a change in specificities (Geha et al., 2000) as was observed in rat. Several reports indicate the existence of large differences in specificities among MAOs of the same subtype but from different mammalian species (Glover and Sandler, 1977, Krueger et al., 1995, Egashira et al., 1999, and Inoue et al., 1999). Some oxadiazolones compounds have inhibitory potencies that vary 4 orders of magnitude between rat and bovine MAO B (Krueger et al. 1995) while some antidepressant drugs show a B over A specificity in the mouse and rat MAOs and the reverse specificity in the rat and monkey MAOs (Egashira et al., 1999). These reports and our results suggest that the specificities of rat and human MAOs (and MAOs of other species) may be determined by different amino acids. Indeed, a computer modeling study suggests spatial differences between the binding sites of MAO A and B and that one amino acid can be responsible for the binding of some but not all substrates and inhibitors (Veselovsky et al., 1998). Also, according to a 3- dimentional MAO model (personal communication, Johan Wouters, Belgium) based on the recently crystallized polyamine oxidase (Binda et al., 1999) which shares a 20% amino acid identity with MAO, Phe-208 and Ile-335 in MAO A and their equivalents in MAO B, lie-199 and Tyr-326 (the amino acids which control specificity in rat and 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. human MAOs respectively), have adjacent sidechains within a region close to the isoalloxazine moiety of the FAD (Fig. 15). This suggests that the overall 3-dimentional structure of human and rat MAOs may be similar but specificity may be determined by different amino acids within the binding domain. MAO A and B may have similar amino acids involved in the active site, however small variations in the 3-dimensional structure results in different specificities. The other 4 mutants (A-T245I, B-I236T; and A-D328G, B-G319D) did not result in any marked differences in their specificities when compared to the wild-types. However their kc a l values for both 5-HT and PEA were lower than those o f the parent enzymes. Among them, a large decrease in kC M was observed in the two MAO A mutants, A- T245I and A-D328G. This indicates that these amino acid residues interact either directly or indirectly with the active site. In summary, the present study identifies the corresponding amino acid pair Ile-335 and Tyr-326 as critical for the substrate and inhibitor specificities in human MAO A and B respectively. Thr-245 and Asp-328 in MAO A and Ile-236 and Gly-319 in MAO A may interact with the active site but do not determine specificity. 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CONCLUSION Figure 15 shows a schematic model of the MAO active site. For simplicity, the amino acid numbering was based on MAO B, however the model also applies to MAO A. MAO B is attached to the mitochondrial outer membrane via the 40 amino acids at the C-terminus, however there may be additional interactions of the protein with the membrane. FAD binds to MAO via a single covalent bond at Cys-406 in A and Cys- 397 in B. Non-covalent interaction include Glu-34 (interacts with the ribose), Tyr-44, Thr-45, Lys-296, and Trp-388 (interact with the isoalloxazine moiety of FAD). The disruption of any of these interactions results in the inactivation of the enzyme. The substrate binding site consists of an aromatic sandwich between the aromatic portions of Tyr-398 and Tyr-435, a catalytic Cys-365 residue, and a specificity determining Tyr-326 residue. However the specificity determining residues are different between human and rat MAOs suggesting the existence of interspecies variations in the substrate binding pocket of MAO. Figure 16 shows a 3-dimentional model of MAO based on the crystal structure of polyamine oxidase as a template (personal communication, Johan Wouters, FUNDP, Belgium). Interestingly, this model reveals a close juxtaposition of the sidechains of the specificity determining residues in rat and in human MAOs (Ile-199 and Tyr-326). This suggests a similar substrate binding pocket for all MAOs from different species but a different set of amino acids controlling specificities. 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The identification of the aromatic sandwich of Tyr-398 and Tyr-435 at opposing sides of the substrate binding pocket suggests that substrate binding to MAO occurs in the following fashion: The common aromatic portion of most MAO substrates intercalates between the benzene rings of the two tyrosine residues stabilized by tc-7t interactions on both sides. In this binding scenario, the substrate remains closely associated with the FAD reaction center, and “locked” into the same plane of movement. Our present work characterizing the active site of MAO and the specificity determining residues will aid in the development o f new highly specific inhibitors following a structural approach. As we have demonstrated, mutagenesis of a particular amino acid results in a nonuniform disruption of binding for different substrates and inhibitors. This indicates that different substrates bind to MAO by interacting with a different set of amino acids within the active site. The 3-dimensional model can be used to understand their different binding mechanisms which can be exploited to design highly specific inhibitors. One approach would be to perform point mutations of the residues within the active site and determine how they affect the binding of large number of ligands. This method would allow the mapping of the binding location of each portion of a the substrate or inhibitor molecule which would be used to design a few, rationally designed, compounds for structure-activity relationship (SAR) studies instead of screening a large library of compounds produced via combinatorial chemistry. 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 14. Schematic model o f MAO. . • ............... lLvs296_ \ N; 2 (Thr45l T F ' ' \ HjO OH • • * ♦ \ / Cvs397 I —s Tvr398 hs—(Cvs365Jpo3 fTvr435 • OH N. _.w Glu34P H zN ' • Cytosol h o o c Intermembrane space o f mitochondria Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 15. 3-dimentional model o f MAO. r v Substrate (PEA) S * C397 Y435 W388 Y398 Y326 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. EXPERIMENTAL PROCEDURES Construction o f the baculovirus transfer vector—Full-length human MAO A and B cDNAs were subcloned into the EcoRl site of pVL1392-transfer vector and named pVLl392-hMAOA and pVL1392-hMAOB respectively. The clone was verified for proper insertion by restriction digest analysis. Recombinant MAO A and B encoding virus was produced by homologous recombination and cotransfection of Sf21 insect cells with the transfer vector and the linearized baculovirus DNA via the calcium phosphate method. Recombinant baculovirus was isolated by plaque purification and amplified by infection of insect cells with a multiplicity of infection (MOI)=l. After 4 passes a viral stock with a titer of 1 0 8 pfu/ml was produced. Construction o f the C-terminal truncation mutants- The single or double stop codons were introduced by site directed mutagenesis at positions indicated below directly into pVL!392-hMAOB. The QuickChange site directed mutagenesis kit (Stratagene Cloning System, La Jolla,CA) was used with complimentary oligonucleotide primers according manufacturer procedure. The following 5’-flanking primers were used for the reactions: C511, CGG CTG TTG GCT TGG TGT AAG ACA AAA GGG GGC TAC and GTA GCC CCC TTT TGT GTT ACA GGA AGC CAA GAG CCG; C504, CCA CCA TCT TTT CAT AAA CGG CTC TTG GCT TCC and GGA AGC CAA TAG CCG TTT ATG AAA AGA TGG TGG; C498, 5’-G CTC AGG CTG ATT GGA TAG ACC ACC ATC TTT TCA GC-3’ and GCT GAA AAG ATG GTG GTC TAT CCA ATC AGG CTG AGC; C492, 5’-G CCC TCC GTG CCA GGC TAG CTC AGG CTG 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ATT GG-3’ and 5’-CC AAT CAG CCT GAG £ T £ GCC TGG CAC GGA GGG C-3’; C486. 5’-G GAG AGA CAT TAQ CCC TCC GTG CC-3’ and 5’-GG CAC GGA GGG CTA ATG TCT CTC C-3’; C481, 5’-CAG CCC ATC ACC ACC ACC TAA TAG GAG AGA CAT TTG CCC TCC G-3’ and 5’-C GGA GGG CAA ATG TCT £ IA TTA GGT GGT GGT GAT GGG CGT-3’; C476, 5’-CT GTG GAT GTC CCT GCA CAG I £ A TAA ACC ACC ACC TTT TTG GAG AG-3’ and 5’-CT CTC CAA AAA GGT GGT GGT TTA TCA CTG TGC AGG GAC ATC CAC AG-3’; C467, 5’-C TGG CAG TCA GAA TGA GAG TCT GTG GAT G-3’ and 5’-C ATC CAC AGA CTC TCA TTC TGA CTG CCA G-3’; C417, 5’-GG GTT CTA CGC CAG TGA GTG GAC AGG-3’ and 5’-CCT GTC CAC J£& CTG GCG TAG AAC CC-3’; C397, 5’-G GAG CAG TAC TCT GGG GGC T£A TAG ACA ACT TAT TTC CCC CCT GGG-3’ and 5’-CCC AGG GGG GAA AT A AGT TGT CTA TCA GCC CCC AGA GTA CTG CTC C-3’ (stop codons are underlined). Expression in Sf21 Insect cells- Small scale expression of carboxy-terminal truncation mutants of MAO-B was carried out in adherent culture of Sf21 cells. 150 mm cell culture dishes were seeded with 20xl06 Sf21 cells and recombinant virus added at MOI of 2. The cells were incubated at 27°C for 72-80 hours and then harvested by centrifugation for 10 min at 5,000 x g. SDS-gel electrophoresis and Western Blotting- SDS-PAGE (sodium-dodecyl-sulphate polyacrylamide gel electrophoresis was carried out according method of Laemmli, 1970 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with modifications (Schagger and Jagow, 1987). Whole cell homogenates or proteins at various stages of purification were denatured by boiling for 5 min in sample buffer (60 mM Tris/HCl buffer, pH 6 .8 , containing 2% (w/v) SDS, 100 mM DTT, 10% (v/v) glycerin and 0.1% (w/v) bromphenol blue. The samples were subjected to 0.75 mm gels consisted of 4% staking and 10% separating gels using BIO-RAD Miniprotean gel apparatus. The separation was carried out at constant current at 30V for staking gel and 1 0 0 V for separating gel. Electrotransfer of proteins to nitrocellulose membrane (Bio-Rad) was carried out at 4°C in transfer buffer containing 15.6 mM Tris-base, 100 mM Glycine and 15%(v/v) methanol at constant current of 120 V for 90 min. Immunodetection of MAO-B was performed at ambient room temperature. Nitrocellulose membrane was incubated with blocking buffer contained 3% (w/v) BSA and 0.05% (v/v) Tween 20 in TBS (tris- buffered-saline, 10 mM Tris/Cl buffer pH 7.5 andl50 mM NaCl) for 2 hours. Nitrocellulose membrane was washed 3 times for 10 min in TBS and incubated with rabbit anti-MAO-B serum (diluted I in 2000 in TBS containing 0.5% (w/v) BSA and 0.05% (v/v) Tween 20) overnight. After first antibody incubation the nitrocellulose membrane was rinsed 3 times for 10 min with TBS and incubated for 2 hours with secondary antibody (goat anti rabbit peroxidase labeled antibody) at 1 in 2 0 0 0 dilution in TBS containing 0.5% (w/v) BSA and 0.05% (v/v) Tween 20. Finally, the filter was rinsed 3 times for 10 min with TBS and the membrane was developed with DAB solution (50 ml TBS containing 50 mg DAB and 30 pi of 30% H2O2. After color was developed the reaction was stopped by rinsing the membrane with water. 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Construction o f MAO A-F208I and MAO B-I199F point mutants - The point mutants MAO A-F208I and MAO B-I199F were made on full length human liver MAO A and B cDNAs cloned into the yeast expression vector pYES2 termed A/pYES2 and B/pYES2 respectively. PYES2 contains the URA3 gene which allows for the selection of transfected cells by their ability to grow on Uracil-lacking media. The QuikChange™ site-directed mutagenesis kit was used with the complementary oligos 5'-(684) CCA CTC GGA TAA TCT CTG TCA CC (706)-3’ and 5’ -(706) GG TGA CAG AGA IT A TCC GAG TGG (684)-3' for MAO A-F208I and oligos 5'-(661) CAA CAA GAA T C I TCT CGA CAA C (682)-3’ and 5'-(682) G TTG TCG AGA AGA TTC TTG TTG (661 )-3* for MAO B-I199F (the mutagenic bases are underlined) and following the protocol of the kit except that an annealing temperature of 52°C was used. Construction o f AB/sv-j/jA and BA 1S 0- 2 05B chimeric enzymes -The strategy to construct the chimeric enzymes is shown in Figure 10. Restriction sites were made using the QuikChangen v i site-directed mutagenesis kit as for the point mutants. The mutations were performed on A/pYES2 and B/pYES2 (yeast expression vector containing full length MAO A or MAO B cDNA respectively). A unique BglH restriction site was introduced in A/pYES2 at amino acid 159 using the complementary oligo pair 5'-CAA AAT GAC CAT GAA AGA TCT CAT TGA CAA AAT C-3' and 5’ -GAT TTT GTC AAT GAG ATC TTT CAT GGT CAT TTT G-3'. A unique BglH restriction site 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. corresponding to that introduced in A/pYES2 was introduced in B/pYES2 at amino acid position 150 using the oligo pair 5'-(505) GGG ACA ACA TGA CAA TGA AAG AIC TAC TGG ACA AGC TCT GC (545)-3* and 5'-(545) GCA GAG CTT GTC CAG TAG ATC IT T CAT TGT CAT GTT GTC CC (505)-3\ Finally, a unique MscI restriction site was introduced at amino acid position 205 in B/pYES2 to correspond to the pre-existing unique MscI restriction site at position 214 in MAO A using the complementary oligo pair 5'-(671) CAT CTC GAC AAC AAA TGG IG G CCA GGA GAG GAA ATT TG (708)-3' and 5’ -(708) CAA ATT TCC TCT CCT GGC CAC CAT TTG TTG TCG AGA TG (67l)-3' (The mutagenic bases are underlined). The A/pYES2 and B/pYES2 constructs with the introduced restriction sites were then double digested with BglH and MscI and the large and small fragments were isolated from the agarose gel. The small and large fragments were then cross-ligated to produce the chimeric constructs A B is^uA (amino acid segment 159-214 of MAO A replaced by the corresponding MAO B segment) and BA150-205B. The successful construction of the point mutants and the chimerics was confirmed by dideoxy sequencing analysis (Sanger et al., 1980). Expression o f the mutants A-F208I, B-I199F and chimerics - The mutant and chimeric constructs were transiently transfected into Saccharomyces cervisiae strain INVScl by the lithium acetate method and grown on Synthetic Complete media lacking uracil (SC- URA) to select for positive colonies. Colonies were picked and grown in SC-URA media containing 2% galactose and 4% raffinose to a cell density of 1x10s cells/ml. The 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cultures were spun down at 1 0 0 0 x g for 1 min, dounce homogenized, and the mitochondria isolated by the differential centrifugation method as described (Yaffe, 1991). Expression o f the mutant clones o f Section II, part 1—The wild-type and mutant MAOs were overexpressed in adherent cultures of Sf21 insect cells (Crossen, 1996). 150 mm cell culture dishes were seeded with 20xl06 Sf21 cells and recombinant virus added at a multiplicity of infection of 2. The cells were incubated at 27°C for 72-80 hr and harvested by centrifugation for 10 min at 5000 x g. Cell pellets were homogenized in 5 ml 20 mM sodium phosphate buffer pH 7.4 containing 0.5 mM EDTA and 0.5 mM PMSF using the polytron homogenizer for 15 sec at setting 5. The homogenates were divided into 0.5 ml aliquots and frozen by dipping into liquid nitrogen and stored at - 80°C. Determination o f enzyme concentration to determine the kca t values (Section II, part 3)—The homogenates were separated by SDS-PAGE and stained by Coomassie brilliant blue R-250. A dense band appeared at the expected Mr of MAO and represented >70% of the total protein. The identity o f the band was further confirmed by western analysis using rabbit anti-MAO A and anti-MAO B antibodies coupled to hydrogen peroxidase. Signals were developped using a peroxidase conjugated goat IgG as secondary antibody followed by exposure to DAB and H2O2. A highly purified BSA standard was electrophoresed in parallel with the homogenates at 9 concentration points 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ranging from 0.1 to 8 p.M. The densities of the MAOs and the BSA bands were quantitated using the EagleSight software and the MAO concentrations were calculated according to the linear BSA standard. Determination o f the kinetic constants (Section II, part 3)- The kinetic constants for the oxidation of 5-HT and PEA and the inhibition by clorgyline, deprenyl, Ro 41-1049, and Ro 16-6491 were determined by the radiochemical method as previously described (Wu et al., 1993) using O2 saturated 50 mM Sodium Phosphate buffer. For the Km determination l4C-5-HT and 14C-PEA concentrations ranged from 0.1 to 5 times the Km values which were determined via Eadie-Hofstee plot (v vs. v/[S]). The kcn / values were calculated from the Vm a x values obtained by fitting the [S] vs. activity curve to the Michaelis-Menten equation and the calculated concentration of the enzyme from the quantitation assay. The IC50 values for the irreversible inhibitors clorgyline and deprenyl were determined by preincubating the inhibitor with the homogenate for 30 min at 37°C and assaying for the remaining activity as described above. 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Creator Geha, Rani Maurice (author)

Core Title A mutational analysis on monoamine oxidase

School Graduate School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology and Toxicology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Shih, Jean Chen (committee chair), [illegible] (committee member), Duncan, Roger (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-218267

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