Indian Journal of Chemistry Vol. 45A. January 2006, pp. 138-145

Topology of human methionine S-adenosyltransferase

Santosh A Khedkar, Alpeshkumar K Malde & Evans C Coutinho* Department of Pharmaceutical Chemistry, Bombay College of Pharmacy

Kalina, Santacruz (E), Mumbai 400 098, India Emai l: evans@bcpindia.org

Received 1 Novelllber 2004; revised 13 November 2005

Insights into the three-dimensional (3D) structure of the enzyme, Methionine S-adenosyltransferase (MAT). can throw light on its role in humans and help in evolving se lectivity attributes of inhibitors targeted at bacterial MAT. We report here a 30-model of human MAT, using the X-ray structure of MAT from the rat as a template, by comparative protein modeling principles. The resulting model has the COITect stereochemistry as gauged from the Ramachandran plot and good 30-structure compatibility as assessed by the Profiles-3D score. The structurally and functiona ll y important residues (active site ) of human MAT have been identified based on information in the rat MAT crystal structure and the point mutation data reported for human MAT. The homology model does conserve the topological and active site features of the MAT family of proteins. However, there exist some differences in the molecular electrostatic potentials (MEP) of MAT from humans and M. tuberculosis. These differences provide a scope for achieving selectivity and specificity of mycobacterium-MAT inhibition over human MAT.

Methionine S-adenosyltransferase (MAT) is an enzyme involved in the biosynthesis of S­adenosylmethionine (AdoMet), which is the principal biological methyl donor and the ultimate source of the propylamine moiety in polyamine biosynthesis. MAT is highly conserved in several pathological microorganisms as well as in humans. Its presence in humans has been an obstacle in the development of antibacterials targeted at the MAT enzyme. MAT is one of 256 enzymes deemed necessary for life l

-3

. It catalyzes the formation of S-adenosylmethionine (Ado Met) using L-methionine and A TP as substrates (Scheme 1). Analysis of the genome sequences of archaea, eubacteria, fungi, plants, and animals has revealed that AdoMet is the principal biological methyl donor and the ultimate source of the propylamine moiety in polyamine biosynthesis4

. The number of reactions that involve AdoMet has been calculated to be as large as those using ATP. This might be the reason why MAT is highly conserved during the course of evolution; approximately 30% residues have been found to be identical in all species.

The presence of a well-conserved enzyme in several pathological microorganisms is a requirement for developing broad-spectrum antibacterials. For this reason, several researchers have considered inhibition of MATas one of several strategies to develop antimicrobial agents against Plasmodiumjalciparum5

,

Mycobacterium tuberculosis4, Pseudomonas

auregenosa6 and Leishmania donovalZi7. A number of

inhibitors of MAT of these species are already reported8

-11

• The crucial role of MAT, both in the active and dormant (chronic) phases of the mycobacterial life cycle, has attracted the attention of TB researchers, to explore it as a potential target for the development of antituberculosis drugs.

Three different forms of MAT (MAT T, MAT II , and MAT III) have been identified in mammalian tissues that are the products of two different genes, MATlA and MAT2A. MAT II is expressed from the MA T2A gene in all mammalian tissues, whereas the MAT1 A gene is expressed only in the liver of adults. The MATIA gene encodes a 395 amino acid catalytic subunit that organizes into dimers (MAT III) and tetramers (MAT I). Under normal conditions, the contribution of MAT II to the hepatic metabolism of methionine is negligible, due to the small amounts of this enzyme expressed in adult human liver as compared to MAT TlIII (referred to as hMA T henceforth in the paper). It is known that the disturbances in the biochemical pathway leading to the synthesis of AdoMet, as catalyzed by MAT, cause demyelination of nerves in the brain, cirrhosis in the Ii ver and formation of tumors 12.

The presence of MAT enzyme in humans has been an obstacle for the development of antibacterial drugs ,

KHEDKAR et al.: TOPOLOGY OF HUMAN METHIONINE S-ADENOSYL TRANSFERASE 139

~N~ 0 0 O '

H N NOli II II ~ '-'::: ~-P-O-P-O-P-OH I I I I

N .....:;N OH OH OH '-..?' HO OH

ATP

. MAT ~ o

Methionine

N~ NH]

H'N~N\°Y"'f~OH 000 II II II

+ HO-P-O-P-O-P-OH I I I

N.....:;N K CH3 0 '-..?' HO OH

OH OH OH

AdoMet

Methyl Donor o 0 II II

HO-P-O-P-OH I I

OH OH

o II

+ HO-P-OH I

OH

Scheme 1

since non-selective inhibition of MAT may lead to complications in humans from AdoMet deficiency. However, the sequence identity between mammalian and bacterial MAT is relatively low, compared to that seen within mammalian or within bacterial MAT enzymes. These differences may be exploited to gain selectivity/specificity over mammalian MAT using molecular modeling approaches. The catalytic activity of an enzyme largely depends on its conformation in the biological system. A vision of the 3D-structure of this enzyme could give insights into the Jigand­enzyme interactions in humans and bacteria that could be used to develop selectivity into lead molecules for these targets. Comparative (or Homology) modeling can be used to significant advantage in the identification and validation of drug targets, as well as for the identification and optimization of lead compounds '3 . We present here a homology model of hMAT using the X-ray crystal structure of MAT from the rat (rMA T) as the reference protein.

Methodology All computations and molecular modeling of

hMA T were carried out on a Silicon Graphics 02 workstation (RSOOO MIPS processor) using the INSIGHT II molecular modeling package (Accelrys Inc., USA)'4. The HOMOLOGY program (Accelrys Inc., USA) was used for comparative protein modeling. The amino acid sequence of hMAT was

obtained from the NCB! protein database 's (GenBank accession no.: 417297). The PSI-BLAST 16 algorithm was used to identify homologous structures for hMAT by searching the structural database of protein sequences in the Protein Data Bank (PDB)I7.

Sequence alignment The crystal structure of MAT from the rat

(rMAT)'8.'9 was selected as a template for homology modeling of the hMAT enzyme, as their sequences share nearly 89% of amino acid identity. The sequence of hMAT was then aligned with the rMAT sequence (extracted from its crystal structure IQM4)'8 using pairwise sequence alignment and further fine­tuned manually, which is shown in Fig.!.

Loop and N-terminal modeling A variable region spanning D 116 to E 127.

corresponding to the active site mobile loop (vide infra) has little sequence identity with the corresponding region in the reference protein. It was modeled with the de novo Loop Generate algorithm20

,

since no similar sequences were identified from the PDB database by the Loop Search algorithm21 . The N-terminal residues M 1 to V 17 were placed in an extended conformation using the End Repair command in the HOMOLOGY module. These two regions, the active site mobile loop and the N­terminal, were finally refined using a simulated annealing (SA) protocol, after the 'side chain rotamer

140

Rat Hliv Htb

INDIAN J CHEM, SEC A, JANUARY 2006

17: GAFHFTSESVGEGHP DKICDQISDAVLDAHLKQDPNAKVACETVCKT 13: LSEGVFHFTSESVGEGHP DKICDQISDAVLDAHLKQDPNAKVACETVCKT 01: HSEKGRLFTSESVTEGHP DKICDAISDSVLDALLAADPRSRVAVETLVTT

Rat 64: GMVLLCGEITSHAHIDYQRVVRDTIKH---IGYDDSAKGFDFKTCNVLVA Hliv 63: GHVLLCGEITSKAHVDYQRVVRDTIKH---IGYDDSAKGFDFKTCNVLVA Htb 51: GQVHVVGEVTTSAKEAFADITNTVRARILEIGYDSSDKGFDGATCGVNIG

Rat 111: LEQQSP-----I------ED---------VGAGDQGLHFGYATDETEECH Hliv 110: LEQQSPDIAQCVHLDRNEED---------VGAGDQGLHFGYATDETEECH Htb 101: IGAQSPDIAQGVDTAHEARVEGAADPLDSQGAGDQGLHFGYAINATPELH

Rat 152: PLTIVLAHKLNTRHADLRRSGVLPWLRPDSKTQVTVQYVQDNGAVIPVRV Hliv 151: PLTIILAHKLNARHADLRRSGLLPWLRPDSKTQVTVQYHQDNGAVIPVRI Htb 151: PLPIALAHRLSRRLTEVRKNGVLPYLRPDGKTQVTIAY--EDNVPV--RL

* * ** * * Rat 202: HTIVISVQHNEDITLEAHREALKEQVlKAVVPA-----KYLDEDTIYHLQ Hliv 201: HTIVISVQHNEDITLEEHRRALKEQVlRAVVPA-----KYLDEDTVYHLQ Htb 197: DTVVISTQHAADIDLEKTLDPDlREKVLNTVLDDLAHETLDASTVRVLVN

Rat 247: PSGRFVIGGPQGDAGVTGRKIIVDTYGGWGAHGGGAFSGKD YTKVDRSAA Hliv 246: PSGRFVIGGPQGDAGVTGRKIIVDTYGGWGAHGGGAFSGKD YTKVDRSAA Htb 247: PTGKFVLGGPUGDAGL TGRKIIVDTYGGWARHGGGAFSGKD PSKVDRSAA

* * ** *** ** * ************ Rat 297: YAARWVAKSLVKAGLCRRVLVQVSYAIGVAEPLSISIFTYGTSKKTEREL Hliv 296: YAARWVAKSLVKAGLCRRVLVQVSYAIGVAEPLSISIFTYGTSQKTEREL Htb 297: YAHRWVAKNVVAAGLAERVEVQVAYAIGKAAPVGLFVETFGTETEDPVKI

Rat 347: LEVVNKNFDLRPGVIVRDLDLKKPIYQKTACYGHFGRSEF--PWEVPKKL Hliv 346: LDVVH-NFDLRPGVIVRDLDLKKPIYQKTACYGHFGRSEF--PWEVPRKL Htb 347: EKAIGEVFDLRPGAIIRDLNLLRPIYAPTAAYGHFGRTDVELPWEQLDKV

***** * * *** ** ****** *** *

Fig. I- Sequence a lignment of MAT enzymes from Rattlls l10rvegiclis (Ratl, human li ver (Hliv ) and M. tuiJerculosis (Mtb) based on . secondary structure and sequence homo logy. The identical residues in a ll aligned sequences are indicated with an asteri sk (* ), acti ve s ite

res idues colored as magenta indicate methion ine binding site, blue as ATP binding site, and red as phosphate binding resid ues. The '-' indi cates gap ill the a li gnment whereas T indicate gaps in crystal structure due to low electro n density.

search' and 'splice-repair' procedures as described in the following sections .

Side chain rotamel' search and splice repair

The Rotamer Search routine in the HOMOLOGY modu le was used to explore the possible range of stable conformations for a certain residue's side chain or those of a set of closely interacting residues, The Auto Rotamer method was used and any possible steric clashes were monitored and relieved appropriately. With the Splice Repair function, the junctions or splice points of segments taken from different templates were smoothened out. This involved an energy minimization procedure with the consistent valence force field (CVFF/2 as implemented in the Discover program (v 98, Accelrys Inc. , USA). The optimization of the splice regions was can'ied out with a combination of steepest descents and conjugate gradients, to a convergence

criterion of 0.001 kcal/mole/A as the deri vati ve.

Simulated annealing and restrained minimizations

maxImum

The structures of the N-tenninal (M I to V 17) and the "active mobile loop" (0 11 6 to E127) residues were refined by an initial minimi zation (steepest descents 20,000 steps; conjugate gradients 10,000 steps) followed by simulated annealing (SA) where all degrees of freedom for these two regions were allowed to relax, but the heavy atoms of all other residues were held rigid. The protocol used for SA involved a slow heating to 600 K in steps of 100 K, followed by slow cooling to 300 K. for a period of 25 picoseconds at every temperature step. The lowest energy structure from the 300 K trajectory was then subjected to a final round of minimization. with all heavy atoms tethered by a force constant of 100 kcal/mole/N. The minimization was done using 10,000 steps each of steepest descents and conjugate gradients.

KHEDKAR el al.: TOPOLOGY OF HUMAN METHIONINE S-ADENOSYLTRANSFERASE 141

The homology model of Mycobacterium tuberculosii3 was generated using the same protocol as described for hMA T with both E. coli and rat MATs, as the reference proteins.

Validation of the hMAT 3D-model The bond lengths, bond angles, torsions and

chirality of the Ca atoms in the model structure were analyzed with the ProStat module (Accelrys Inc. , USA). The accuracy and validity of the model was tested with Profiles-3D (Accelrys Inc., USA)24, which calculates a 3D to 1D compatibility score, and graphically portrays the properly folded and mi sfolded region(s) in the protein structure by performing an Eisenberg analysis25.26 of the model.

Molecular electrostatic potential (MEP) The electrostatic interaction is a crucial part of the

non-bonded interaction energy between molecules . The electrostatic potential on a molecul ar surface can be used to vi sually compare different molecules. It is also useful for guiding docking studies. The molec ular electrostatic potential on a protein surface can be used to find sites that act attractively on li gands by matching 'J pposite electrostatics . MEP is calculated by the following equation:

N EP(i)= I

q. J

j=l r .. IJ

where, EP( i) is the electrostatic potential at the surface point i due to atom j having the partial charge qj and separated by distance r ij. The electrostatic potential (EP) on the surface is generally colored according to the sign and magnitude of the potential. The color ramp for EP ranges from red (most positive) to purple (most negative) .

All MEP calculations and visualization were carried out using the MOLCAD program implemented in the SYBYL molecular modeling package (Tripos Inc. , USA)2C,. The hydrogens to the ionizable groups in side chains of both hMAT and Mtb-MAT22

homology models were added using the Insight /1 Biopolymer program at pH 7.4, resulting in a + 1 charge for arginines and lysines, and -I charge for the aspartates and glutamates . The homology models of hMAT and Mtb-MAT, were superimposed onto the rat MA T crystal structure (1 QM4) and the corresponding residues within a 7 A radius of the inhibitor (L-cis-AMB) in the lQM4 crystal structure

Table I- A comparison of the corresponding residues ill the active sites of rat liver, human liver and M. IUhercu/o.l'is MAT enzy mes, within a 7 A radius from the inhibitor in rat crystal structure

Rat liver HUlllan Mtn

Substrate / inhibitor binding residues

Val26 Val25 Va l 13 Gly29 Gly2S Gly l6 Hi s30 Hi s29 Hi s l7 Pro3 1 Pro30 ProlS

Asp ISO Aspl79 Aspl79

SerlSI * Ser ISO* Gly ISO' Lys l 82 Lys l S I Lys lS I Ser207 Ser206 Ser202

Va120S* Val20Y Thr203'1' Pro247 Pro246 Pl'o247

Ser24S* Ser247 * 1'111'248'" Gly249 Gly248 G ly249

Arg250* Arg249 * Lys250* Phe251 Phe250 Phe25 I Val252 Val25l Val252 Tl e253 lle252 lle253

Gly254 Gly253 Gly254 Gl y255 Gly254 G ly25S Pro256 Pro255 Pro256

Gln257* Gln256* Met257* Gly258 Gly257 G ly258 Asp259 Asp2S8 Asp259

ATP binding residues

Lys54* LysS3* .Arg4 1 .;.

Val55 Val54 Va l42 Ala56 Ala55 Ala43 G lu71 Glu70 GluSR Ilen* IIe7 1 " Va159* Thr73 Thrn T hr60 Lys286 Lys285 Lys2R6 Ly s290 Lys289 Lys290

*Mutated residues in three MATs

(Table I, residues shown in magenta in Fig. I) were identified. The Gasteiger-HUckel charges were assigned to the atoms of both structures (Mtb and human MAT models). The electron density isosurfaces for both proteins were calculated with a cut value of 0.003 and step width of O.S A, over which the MEP surfaces were generated and visualized.

Results and Discussion The various aspects that affect the quality of the

homology model such as overall sequence identity. extent of binding site conservation, size and location of insertions and deletions and resolution 28 were all actively considered while choosing the template structure for the co;mpar:...:ive modeling of hMA T. A

142 INDIAN J CH EM, SEC A, JANUARY 2006

PSI-BLAST I6 search of the PDB database revealed 12 crys tal structures of the MAT enzyme as a native or co-crystalli zed with either a substrate or an inhibitor. Of these, 5 structures were from the common rat (Rattus I/orvegicus; POB codes: 1 QM4, 1090, 1092, 1093, 109T)i81 'J and 7 structures from E. coli (PDB codes : IMXA, lMXB, IMXC, IXRA, IXRB , IXRC, and I FUG)c(J.30. The crystal structure (lQM4) 18 with

the hi ghes t resolution (2.66 A) and highest sequence identity was chosen as a templ ate for the alignment. T he coordinates of the side chains and the backbone atoms were copied to the target sequence onl y if, identi cal amino acids were found at corresponding pos itions in the sequence; for 'similar' but no t 'identica l' amino acids, only the common side chain atoms were copied, while fo r the rest, amino acid confo rmati ons from the library were used . After ass igning the coordinates from the reference structure to the target sequence, loops were modeled by searching for similar sequences in the PDB database, and JI1 cases where no 'hits' were obtained, coordinates were assigned using the Loop Generate algorithm. The N-terminal region was placed in an extended conformation . Then, every conformation of the side chain of amino acids both in the conserved and vari able regions was explored, and those with minimum steric clashes were re tained. After modeling the vari able regions with simul ated annealing, the co mpl ete structure was fin ally refined by energy minimization to remove any steric clashes of the side chains with each other and/or with backbone atoms.

The <p and \jf dihedral ang les in the model structure lie within the allowed regions of the Ramachandran plot. T he overall 3D-structure compatibility (Proj iles-3D) score was 167, which is far higher than the threshold score of 81 for a protein of this SIze, ex press ing a strong confidence in the 3D model.

Mobile loop The "acti ve site mobil e loop" (spanning 011 6 to

E I27) 18.1<).2<)-32, control s the entry of the

substrate/inhibitor into the ac ti ve site. Mol ecular dynami cs simulations of thi s loop show that it can adopt a wide range of low energy conformations, which accounts for its flexibility and hence absence in the X-ray diffraction electron density mapsI8.19.29.30.

An alignment of a large vari ety of MATs from bacteri a, yeast, plants, Drosophila, mouse, rat and human li ver and mouse, rat and human kidney shows that cys teine is present at position ] 2 1 only in the li ver enzy me, which is repl aced most often by glycine

Fig. 2- Structural topology o f hMAT obt<J ined by co mparati ve modeling. Helices (H) <Jre co lored magenta and ~- strands (8) as yellow. Nand C indi cate the N- tennin al and C-termin al reg ions of hMAT protein. H's and 8 's are numbered along the amino ac id sequence from the N- to C- terminal.

in others. Cys 121 is located over the active site of the enzyme in the fl ex ible loop. Interesting ly , when cysteine is repl aced by serine (CI 2 IS ) in thi s loop, the enzyme is still active but is now res istant to inactivation by nitric oxide (NO) and reactive oxygen substances (ROS)1 2. This fact coupled wi th the different length of the mobile loop in humans and bacteria could be used to advant~e in conferring some selectivity charac teri stics into inhibitors_

Topology of the hMA T enzyme There are three domains - the N-terminal domain ,

the central domain, and the C-terminaI domain , so termed because of their location in the tertiary structure. Each domain is constituted of two ex-helices and three or four ~- strand s . A long ex-helix , fo llowed

by a pair of anti-parallel ~-strands and a short ex- heli x (e.g ., Hl-~2-~3-H2 in the central domain , Fi g. 2) is a

-common secondary structural motif in each domain _ Correspondingly, the N-terminal and C-termi nal domains are formed by H3 -~6-~7-H4 and H6-~10-

~11-H7 motifs, respecti vely . The strands ~4 and ~8 form oarallel ~-sheets with ~3 and ~7 in the central and N-terminal domains, respectively. On the other hand, ~5 forms an anti parallel sheet with ~ lOin the C-terminal domain. The fourth strand, ~9 in the central domain and ~l in the N-terminal domain fo rm antiparallel sheets with ~2 and ~6 strands, respectively. The "ac ti ve mobile loop" which fo rms a bridge between the central and the C-terminal

KHEDKAR el 01.: TOPOLOGY OF HUMAN METHIONINE S-ADENOSYL TRANSFERASE 143

Fig. 3--The absolute values of the mo lecular e lectrostatic pote ntial (MEP) displayed for the .active s ites o f hMAT (Al and Mtb-MAT (13).

The deep blue co lor indicates the hi ghes t negative potential whereas the most positive (the lowest negative) potential is see n as deep red color. The co lor spectrum show n to the left shows the gradation of e lec trostatic potential in the respec ti ve enzymes . In order to ma ke valid comparisons between fi gures (A) and (B), the electrostatic potentials have been put on the same scale which are show n in (el for hMAT and (D) for Mtb-MAT enzy mes.

domains occupies a large conformational space. A close inspection of the hMAT model reveals that it has all the topological features of this protein family .

The hMA T active site

The residues forming the active site of hMAT have a good correspondence to other MAT enzymes, except for a few mutations (Fig. 1, Table I). The hMA T active site is located in a broad cavity composed of the dimer interface involving residues from both subunits. The A TP-binding region is composed of G(l31 )AGDQG(136) residues (colored blue in Fig. 1), while the segment H(277)GGGAFSGKD(286) is the phosphate-binding

. P-loop (colored red in Fig. I). The methionine­binding region (coloured magenta in Fig. 1) is formed by residues F(250)VIGGPQGDA(259) in the loop which connects the [38-strand with [39 as seen in Fig. 2. The overall acti ve site of hMA T is conserved with respect to rMA T, but there are significant mutations

when compared to bacteri al MATs, specifically, Mtb­MAT. It is important to mention that the A TP and methionine binding sites lie in different chains of the dimer, and together form the full active site where the synthesis of AdoMet (Scheme 1)" takes place. It is known that potassium and magnesium ions confer rig idity to the active site by forming a salt bridge between residues of the two-monomer chains. A magnesium ion coordinates the carboxylate oxygen atom of the inhibitor (L-cis-AMB) and the s ide chain carboxylate oxygen atom of Asp 179 18

.1'). The sile­directed mutagenesi s studies carried out for rM AT have led to the conclusion that Phe251, Asp 180, and Lys 182, are critical for AdoMet synthesis either by binding methionine or possibly by being involved in subsequent steps of the mechanism. These conclusions were arrived at from the fact that th eir mutations result in an absolute loss of MAT activity when compared to the native enzyme 1R

.19

. The

144 INDIAN J C HEM, SEC A, JAN UARY 2006

corresponding residues in the active site In rMAT, hMAT and Mtb-MAT are listed in Table 1.

Molecular electrostatic potential (MEP) The MEP is a popular indicator of electrophilic and

nucleophilic centers, which governs the strength of bonds, the strength of non-bonded interactions and molecular reactivity. It affec ts the strength of the interaction of the ligand with the receptor protein . Bhattacharrjee and Karle have used the MEP to rel ate the antimalarial potency of carbinolamine analogs34

and neurotoxicity of artemisinin analogs3s. In case of

a li gand-protein interaction, at the active site, the ligand experiences a unique environment in terms of electrostatic, steric and hydrophobic properties. Variations in these properties in the active sites of di fferent proteins can contribute to selecti ve/specific or tighter binding of the li gand to enzy me proteins. Thus, a comparison of the MEPs of hMAT and Mtb­MAT23 is one-step towards understanding select~vity in MAT inhibition.

The MEP surfaces are shown with absolute values of the electrostatic potential for hMA T (+ 116 to -332; Fig. 3A) and Mtb-MAT (-2 to -458; Fig. 3B). Since the range of potentials in the two proteins differ greatl y, meaningful comparisons can only be made when the electrostatic potenti al of the two proteins are placed on the same scale + 116 to -458. This is now shown in Figs 3C and 3D for hMAT and Mtb-MAT, respectively. As is evident in Figs 3C and 3D, the electrostatic potential covering the active site residues have sharply differing values, with the active site of Mtb being more deeply negative. Within, hMAT itself. the surface around Asp 179, Gly2S7 and Asp2S8 residues has a relati vely more electronegative potential when compared with other regions in the ac tive site. This same contrasting feature is visible for the region encompassing residues Asp 179, Gly16 and His 17 in the active site of Mtb-MA T. This means that the two proteins will bind inhibitors in the same relative sense I 8

.19

, but the binding affinities of a given inhibitor will not be the same for the two proteins, due to the diffe rences in the absolute values of the electrostatic potential. Further, the three-fold difference in the value of the potential at Asp179 in hMAT (-113) and Mtb-MAT (- 382) will have a deep impact on the selective binding of methionine analogs via coordination of the Mg2+ ion IH. 19.

Conclusions Although, the sequences of the MAT enzymes are

well conserved along the evolution, it is possible that

differences in the active site (as evident in the MEPs) may account for the environmental particul ariti es of each species. One can gainfully use the differences in the electrostatic potentials of hMAT and Mtb-MA T to design inhibitors that are specific and selective against Mtb-MAT.

Acknowledgements This work was made possible by a grant (F. No.

8022/RlD/NPROJ/RPS-SI2003-04) from the All Indi a Council of Technical Education (AICTE), New Delhi and the Department of Science and Technology (DST), through their FIST program (SR/FST/LSI-08312003) to E. Coutinho. SAK thanks the Lady Tata Memorial Trust, Mumbai , and AKM the University Grants Commission (UGC, New Delhi) for fina ncial support.

References I Tabor C W & Tabor H. Adv EllzYl1wl, 56 (J 984) 25 1. 2 Kotb M & Geller A M. Plwrmacol Ther. 59 (1993) 125. 3 Mato J M, Alvarz L. O ri tz P & Pajares M A, Plwrl7/ocol Ther.

73 (1997) 265. 4 Berger B J & Knodel M H, BMC Microbial, 3 (2003) 12. 5 Chiang P K. Chamberlin M E. Nicholson D. Soubcs S, Su X­

Z, Subramanian G, Lanar D E, Prigge S T. Scov ill J P. Miller L H & Chou J Y, Biochem J, 344 (1999) 571.

6 Stover C K. Pham X.-Q T. Erwin A L. Mi zoguchi S D. Warrener P, Hickey M J. Brinkman F S L. Hufnagle W O. Kowalik D J, Lagrou M, Garber R L, Go ltry L. Tolentino E. Westbrook-Wadman S, Yuan Y, Brody L L. C\),liter S N. Folger K R, Kas A, Larbig K, Lim R M. Smith K A, Spencer D H, Wong G K-S, Wu Z, Paulsen I T, Reizer 1. Saier M H, Hancock R E W. Lory S & Olson M V. Natu re. 406 (2000) 959.

7 Perez-Peltejo Y, Reguera R M. Villa H, Garcia-Estrada C. Balana-Fouce R, Pajares M A & Ordonez D, Ellr J Biochelll. 270 (2003 ) 28.

8 Surfin J R. Cou lter A W & Talalay P. Mol Plwnnacol. 15 (1979) 661.

9 Federici M M & Lotspeich F 1. Biochelll PharnJ({col, 28 (1979) 1689.

10 HulbertP B, Mol Phamwcol, 10 (1974) 3 15. II Lombardini J B & SufI'i n J R, Biochelll PhoJ7l1acol, 32, ( 1983)

489. 12 Mato J M , COlTales F J, Lu S C & Avila M A, The FASEB J.

16 (2002) 15. 13 Wieman H, Tondcl K, Anderssen E & Hilgenfe ld R. Milli -Rel'

Med Chell1, 4 (2004) 793 . 14 Insighlll. version 98. Accelrys Inc .. San Diego, CA. USA. 15 http://www.ncbi.nlm.nih.gov/protein. 16 Altschul S F, Madden T L. Schaffer A A. Zhang 1. Zhang Z.

Miller W & Lipman D J. Nucleic Acids Res, 25 ( 1997) 3389. 17 Berman H M, Westbrook J, Feng Z. Gilliland G. Bhat T N.

Weissig H, Shindyalov I N & Bourne P E. Nucleic Acids Res, 28 (2000) 235.

18 Gonzales B, Pajares M A, Hermosa J A, A lvarez L. Garrido F. Sufrin J R & Sanz-Aparic io J. J Mol Bioi, 300 (2000) 363.

KHEDKAR et al.: TOPOLOGY OF HUMAN METHIONINE S-ADENOSYL TRANSFERASE 145

19 Gonzales B. Pajares M A, Hermoso J A, Guillerm D, Guillerm G & Sanz-Aparicio J. J Mol Bioi. 331 (2003) 407.

20 Shenkin P S. Yarmush D L. Fine R M. Wang H & Levinthal C. Biopo[vlIlers. 26 (1987) 2053.

21 Hobohm U. Scharf M. Schneider R & Sander C , Prolein Science, I (1992) 409.

22 Dauber-Osguthorpe p , Roberts V A, Osguthorpe D 1, Wolff 1. Genes t M & Hagler A T, Proteins, 4 (1988) 31.

23 Khedkar S A, Malde A K & Coutinho E C. J Mol Graph Model. 23 (2005) 355.

24 Luthy R. Bowie 1 U & Eisenberg D, Nature, 356 (1992) 83. 2S Gribskov M. Mclachlan A D & Eisenberg D. Proc Natl Acad

Sci USA, 84 (1987) 4355. 26 Gribskov M. Luthy R & Eisenberg D, Metll Enzymol, 183

(1990) 146.

27 Syby/, version 6.7, Tripos Inc., St. Louis, MO, USA. 28 Diller D 1 & Li R, J Med Chem, 46 (2003) 4638. 29 Takusagawa F, Kamitori S & Markham G D, BiochelllisflT .

35 (1996),2586. 30 Takusagawa F, Kamitori S, Misaki S & Markh am G D. J

Biol C/wn. 271 ( 1996) 136. 3 1 Fu Z, Hu Y. Markham G D & Takusagawa F, J Biolllo/

Struct DYIl. 13 (1996) 727. 32 Taylor 1 C & Markham G D. Arch Biochelll Biophys, 4 15

(2003) 164. 33 Parry R & Minta A. J Alii Chelll Soc. 104 (1982) 87 1. 34 Bhattacharrjee A K & Karle 1 M, Bioorg Med Chelll .' 6

(1998) 1927. 35 Bhattacharrjee A K & Karle J M, Chem Res Toxico/ . 12

(1999) 422.