Structural and Biochemical Studies of Serine Acetyltransferase Reveal Why the Parasite Entamoeba histolytica Cannot Form a Cysteine Synthase Complex

Abstract

Cysteine (Cys) plays a major role in growth and survival of the human parasite Entamoeba histolytica. We report here the crystal structure of serine acetyltransferase (SAT) isoform 1, a cysteine biosynthetic pathway enzyme from E. histolytica (EhSAT1) at 1.77 Å, in complex with its substrate serine (Ser) at 1.59 Å and inhibitor Cys at 1.78 Å resolution. EhSAT1 exists as a trimer both in solution as well as in crystal structure, unlike hexamers formed by other known SATs. The difference in oligomeric state is due to the N-terminal region of the EhSAT1, which has very low sequence similarity to known structures, also differs in orientation and charge distribution. The Ser and Cys bind to the same site, confirming that Cys is a competitive inhibitor of Ser. The disordered C-terminal region and the loop near the active site are responsible for solvent-accessible acetyl-CoA binding site and, thus, lose inhibition to acetyl-CoA by the feedback inhibitor Cys. Docking and fluorescence studies show that EhSAT1 C-terminal-mimicking peptides can bind to O-acetyl serine sulfhydrylase (EhOASS), whereas native C-terminal peptide does not show any binding. To test further, C-terminal end of EhSAT1 was mutated and found that it inhibits EhOASS, confirming modified EhSAT1 can bind to EhOASS. The apparent inability of EhSAT1 to form a hexamer and differences in the C-terminal region are likely to be the major reasons for the lack of formation of the large cysteine synthase complex and loss of a complex regulatory mechanism in E. histolytica. Cysteine (Cys) plays a major role in growth and survival of the human parasite Entamoeba histolytica. We report here the crystal structure of serine acetyltransferase (SAT) isoform 1, a cysteine biosynthetic pathway enzyme from E. histolytica (EhSAT1) at 1.77 Å, in complex with its substrate serine (Ser) at 1.59 Å and inhibitor Cys at 1.78 Å resolution. EhSAT1 exists as a trimer both in solution as well as in crystal structure, unlike hexamers formed by other known SATs. The difference in oligomeric state is due to the N-terminal region of the EhSAT1, which has very low sequence similarity to known structures, also differs in orientation and charge distribution. The Ser and Cys bind to the same site, confirming that Cys is a competitive inhibitor of Ser. The disordered C-terminal region and the loop near the active site are responsible for solvent-accessible acetyl-CoA binding site and, thus, lose inhibition to acetyl-CoA by the feedback inhibitor Cys. Docking and fluorescence studies show that EhSAT1 C-terminal-mimicking peptides can bind to O-acetyl serine sulfhydrylase (EhOASS), whereas native C-terminal peptide does not show any binding. To test further, C-terminal end of EhSAT1 was mutated and found that it inhibits EhOASS, confirming modified EhSAT1 can bind to EhOASS. The apparent inability of EhSAT1 to form a hexamer and differences in the C-terminal region are likely to be the major reasons for the lack of formation of the large cysteine synthase complex and loss of a complex regulatory mechanism in E. histolytica. IntroductionIn bacteria and plants, l-cysteine is synthesized from l-Serine by two key enzymes serine acetyltransferase (SAT) 3The abbreviations used are: SAT, serine acetyltransferase; OASS, O-acetyl serine (OAS) sulfhydrylase; DTNB, 5,5′-dithio-bis(2-nitrobenzoic acid); PLP, pyridoxal-5′-phosphate; TNB, 5-thio(2-nitrobenzoate); Ec-, E. coli; Hi-, H. influenzae; Eh, E. histolytica; r.m.s.d., root mean square deviation. and O-acetyl serine sulfhydrylase (OASS). SAT converts l-Serine to O-acetyl serine by transferring an acetyl group from acetyl-CoA. Later, OASS converts the O-acetyl serine (OAS) to l-cysteine by adding a sulfide. This cysteine biosynthetic pathway contributes significantly to the incorporation of inorganic sulfur into organic compounds. SAT is in low abundance compared with OASS and is the rate-limiting component in this pathway (1Smith I.K. Thompson J.F. Biochim. Biophys. Acta. 1971; 227: 288-295Crossref PubMed Scopus (58) Google Scholar).The intracellular level of Cys is maintained by complex kinetics and strictly regulated by two known mechanisms. In the first, SAT is inhibited by Cys through a feedback mechanism. The second mechanism involves association of SAT and OASS to form a cysteine synthase complex. In the complex form, SAT activity increases, whereas OASS activity is reduced (2Saito K. Yokoyama H. Noji M. Murakoshi I. J. Biol. Chem. 1995; 270: 16321-16326Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This results in excess production of OAS, which causes the dissociation of the cysteine synthase complex and subsequent down-regulation of SAT activity (3Kredich N.M. Becker M.A. Tomkins G.M. J. Biol. Chem. 1969; 244: 2428-2439Abstract Full Text PDF PubMed Google Scholar).The structures of SAT from Escherichia coli (EcSAT) and Haemophilus influenzae (HiSAT) have been reported (4Pye V.E. Tingey A.P. Robson R.L. Moody P.C. J. Biol. Chem. 2004; 279: 40729-40736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 5Gorman J. Shapiro L. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 1600-1605Crossref PubMed Scopus (29) Google Scholar) and also in complex with cysteine and acetyl-CoA (6Olsen L.R. Huang B. Vetting M.W. Roderick S.L. Biochemistry. 2004; 43: 6013-6019Crossref PubMed Scopus (67) Google Scholar). These structures exist as hexamers in the form of dimer of trimers. The solution studies and structures of OASS reveal that it stays as a dimer (7Krishna C. Jain R. Kashav T. Wadhwa D. Alam N. Gourinath S. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2007; 63: 512-515Crossref PubMed Scopus (10) Google Scholar, 8Becker M.A. Kredich N.M. Tomkins G.M. J. Biol. Chem. 1969; 244: 2418-2427Abstract Full Text PDF PubMed Google Scholar, 9Chinthalapudi K. Kumar M. Kumar S. Jain S. Alam N. Gourinath S. Proteins. 2008; 72: 1222-1232Crossref PubMed Scopus (35) Google Scholar). Biochemical and modeling studies indicated that one SAT hexamer and two OASS dimers interact to form the cysteine synthase complex (10Feldman-Salit A. Wirtz M. Hell R. Wade R.C. J. Mol. Biol. 2009; 386: 37-59Crossref PubMed Scopus (65) Google Scholar). The molecular details of the cysteine synthase complex are still not very clear. Various biochemical and structural studies have revealed that C-terminal end of SAT interacts with the active site of OASS (11Zhao C. Moriga Y. Feng B. Kumada Y. Imanaka H. Imamura K. Nakanishi K. Biochem. Biophys. Res. Commun. 2006; 341: 911-916Crossref PubMed Scopus (42) Google Scholar, 27Denk D. Böck A. J. Gen. Microbiol. 1987; 133: 515-525PubMed Google Scholar).Entamoeba histolytica is the enteric protozoan parasite that causes amoebic colitis and extra-intestinal abscesses in approximately 50 million people and kills about 70,000 each year (12WHO/PAHO/UNESCO Epidemiol. Bull. 1997; 18: 13-14Google Scholar). Cysteine was reported as the major thiol in E. histolytica (13Nozaki T. Asai T. Kobayashi S. Ikegami F. Noji M. Saito K. Takeuchi T. Mol. Biochem. Parasitol. 1998; 97: 33-44Crossref PubMed Scopus (82) Google Scholar) and is assumed to play a major role in oxidative stress defense mechanisms in this glutathione-deficient organism. Cysteine has also been shown to be necessary for survival, growth, elongation, and attachment to matrix (14Nozaki T. Asai T. Sanchez L.B. Kobayashi S. Nakazawa M. Takeuchi T. J. Biol. Chem. 1999; 274: 32445-32452Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 15Nozaki T. Ali V. Tokoro M. Adv. Parasitol. 2005; 60: 1-99Crossref PubMed Scopus (94) Google Scholar). The cysteine biosynthetic pathway could be crucial target to develop new chemotherapeutics (16Agarwal S.M. Jain R. Bhattacharya A. Azam A. Int. J. Parasitol. 2008; 38: 137-141Crossref PubMed Scopus (37) Google Scholar). Similar to other bacteria and plants, l-cysteine is synthesized by serine acetyltransferase (EhSAT) and O-acetyl serine sulfhydrylase (EhOASS) in E. histolytica. But unlike in other bacteria and plants, EhSAT and EhOASS do not interact with each other and do not form the cysteine synthase complex (14Nozaki T. Asai T. Sanchez L.B. Kobayashi S. Nakazawa M. Takeuchi T. J. Biol. Chem. 1999; 274: 32445-32452Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar).The fact that the E. histolytica genome encodes three forms of SAT suggests the importance of this enzyme for its survival. The EhSAT1 and EhSAT2 isoforms are very similar (78% identity), whereas the EhSAT3 isoform has very low sequence homology with EhSAT1 and EhSAT2 (48% identity). These three isoforms show differences in sensitivity to the feedback inhibitor l-cysteine (17Hussain S. Ali V. Jeelani G. Nozaki T. Mol. Biochem. Parasitol. 2009; 163: 39-47Crossref PubMed Scopus (32) Google Scholar). Till now there is no structural information available as to how sensitivity for feedback inhibition of cysteine is lost for some mutants/isoforms and why cysteine synthase complex does not form in E. histolytica.Here for the first time we report the structure of EhSAT at 1.77 Å resolution as well as its structure bound with its substrate serine at 1.59 Å resolution and its inhibitor cysteine at 1.78 Å resolution. EhSAT1 in these structures is trimeric rather than hexameric. The structures with Ser and Cys indicate how Cys can be a competitive inhibitor with Ser and does not inhibit acetyl-CoA. The docking and fluorescence binding studies with EhOASS clearly show that if the EhSAT1 had different C-terminal sequence, EhSAT1 and EhOASS could interact with each other. The C-terminal mutant of EhSAT1 does inhibit EhOASS, confirming our docking studies. These new structural features and biochemical studies suggest abolition of cysteine synthase complex formation to ensure ample supply of cysteine for the survival of the organism.DISCUSSIONSequence comparison of EhSAT1 with other SATs shows very high diversity in the N-terminal domain region (supplemental Fig. 2). The structure of N-terminal domain is helix-rich, with eight helices, which is similar to and the orientation of the N-terminal domain is different in comparison with details The C-terminal domain sequence and structure is well with a This domain also the active site, which is near the The loop near the is and three could not be the to The active site in the native structure is with a with and a with SAT is the first time any serine acetyltransferase was as a trimer in both solution as well as in the crystal and as hexamers in crystal structures (4Pye V.E. Tingey A.P. Robson R.L. Moody P.C. J. Biol. Chem. 2004; 279: 40729-40736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 5Gorman J. Shapiro L. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 1600-1605Crossref PubMed Scopus (29) Google Scholar) as well as in Moody P.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google two interact with each other in the of each of EhSAT1 is similar to each of of Å for and of Å for the of N-terminal domain and C-terminal The structure of the C-terminal domain is similar in the different this region is also and the active are two C-terminal of to three active for each In about of the N-terminal domain of EhSAT1 are different compared with and The major difference at about and and Fig. 2). In and a loop these helices, are about to one In EhSAT1, and are with a near The N-terminal region also differs due to the of in EhSAT1 compared with (supplemental Fig. 2). In EhSAT1, and are in to each other and can form a This of is not in other as these are not N-terminal domain to the oligomeric state of The N-terminal domain of EhSAT with and is and Å, the N-terminal region of EhSAT1 has very low sequence similarity with other SATs. The first of the N-terminal domain have no sequence similarity with and there is an of about eight in the of N-terminal domain of the of is the same in the N-terminal domain of SAT structures, the orientation of first is In to and the N-terminal from the trimeric of the form a the is in EhSAT1 in to the of the trimer in which to a of the same in EhSAT1 is these structural differences be the that EhSAT1 does not form a charge the of and EhSAT1 from the N-terminal of and have compared with EhSAT1 and be responsible for two of and to interact and form in loop near the which is of the active site, has an to EhSAT1 as compared with and This could the activity of the enzyme and the with the C-terminal In the complex structure, of near the active site, forms a with of the C-terminal region of the (supplemental Fig. In this structure, the C-terminal region is well to and are disordered at the C-terminal In complex structure, interacts with and, thus, interact with the C-terminal due to the C-terminal region was and could not be in the structure (6Olsen L.R. Huang B. Vetting M.W. Roderick S.L. Biochemistry. 2004; 43: 6013-6019Crossref PubMed Scopus (67) Google Scholar). But in the of EhSAT1, is in the sequence, and other do not for these This lack of of C-terminal region be responsible for it disordered in EhSAT1 the acetyl-CoA binding site is and in three structures of EhSAT1 the complex of in E. coli SAT to the enzyme to cysteine inhibition D. Böck A. J. Gen. Microbiol. 1987; 133: 515-525PubMed Google Scholar). are be in the necessary for cysteine inhibition sensitivity K. T. T. N. A. Nakanishi K. Biochem. 1999; 63: PubMed Scopus Google Scholar, K. T. T. N. A. Nakanishi K. Biochem. PubMed Scopus (65) Google Scholar). at this is not in EhSAT1, and this region in the structures of EhSAT1 is not and could not be this enzyme was shown to be to cysteine by competitive inhibition (17Hussain S. Ali V. Jeelani G. Nozaki T. Mol. Biochem. Parasitol. 2009; 163: 39-47Crossref PubMed Scopus (32) Google Scholar). In EhSAT1, competitive inhibition by Cys be due to to the binding site and the binding differences Cys and the of it is clearly that in the structure, the C-terminal end of the interacts with the and the acetyl-CoA binding site (6Olsen L.R. Huang B. Vetting M.W. Roderick S.L. Biochemistry. 2004; 43: 6013-6019Crossref PubMed Scopus (67) Google Scholar) (supplemental Fig. thus, complex kinetics in which Cys to with acetyl-CoA N.M. Tomkins G.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) be that the inhibition for is low compared with EhSAT1, as the C-terminal region also be in inhibition the acetyl-CoA binding site, it is not in In the of the inhibition is a of Cys at the active site and binding of the C-terminal region near the active site by the acetyl-CoA binding that the loop of the and the C-terminal end of EhSAT1 are different compared with and are no these in EhSAT1, which in the In the of EhSAT1, the kinetics be competitive inhibition of Cys to Ser and not have any acetyl-CoA and this is clearly in studies reported by (17Hussain S. Ali V. Jeelani G. Nozaki T. Mol. Biochem. Parasitol. 2009; 163: 39-47Crossref PubMed Scopus (32) Google of Cys inhibition (17Hussain S. Ali V. Jeelani G. Nozaki T. Mol. Biochem. Parasitol. 2009; 163: 39-47Crossref PubMed Scopus (32) Google and Tomkins (17Hussain S. Ali V. Jeelani G. Nozaki T. Mol. Biochem. Parasitol. 2009; 163: 39-47Crossref PubMed Scopus (32) Google Scholar) and Biochem. J. PubMed Google Huang B. Roderick S.L. Biochem. Biophys. 2004; PubMed Scopus (29) Google Scholar) in a new EhSAT1 C-terminal with is known from studies E. H. and M. enzymes that the of SAT interacts with the active site of OASS (10Feldman-Salit A. Wirtz M. Hell R. Wade R.C. J. Mol. Biol. 2009; 386: 37-59Crossref PubMed Scopus (65) Google Scholar, C. Moriga Y. Feng B. Kumada Y. Imanaka H. Imamura K. Nakanishi K. Biochem. Biophys. Res. Commun. 2006; 341: 911-916Crossref PubMed Scopus (42) Google is at the C-terminal the EhSAT1 has at the C-terminal it has been reported that it not interact with EhOASS (13Nozaki T. Asai T. Kobayashi S. Ikegami F. Noji M. Saito K. Takeuchi T. Mol. Biochem. Parasitol. 1998; 97: 33-44Crossref PubMed Scopus (82) Google Scholar, T. Asai T. Sanchez L.B. Kobayashi S. Nakazawa M. Takeuchi T. J. Biol. Chem. 1999; 274: 32445-32452Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). To further, we peptide from the EhSAT1 C-terminal sequence and for binding and inhibition studies of EhOASS. The peptide about competitive inhibition not show any in that it does not bind to EhOASS active To the for the loss of these we a of the C-terminal sequence of SAT by at the and docking studies to the active site of studies of SAT C-terminal peptides with EhOASS structure that peptides could have binding than the C-terminal peptides that have binding to EhOASS and for the inhibition activity and binding the EhOASS. and show inhibition and binding the EhOASS compared with be that is the C-terminal sequence of M. The docking studies suggest that the and with the EhOASS active site that if the C-terminal end of EhSAT1 had any of these peptide the EhSAT1 have with EhOASS to form a complex. But this complex not be a as in other cysteine synthase the SAT in other is a hexamer and EhSAT1 is a we the of EhOASS structure that the of EhOASS be responsible for loss of EhOASS and EhSAT1 K. Kumar M. Kumar S. Jain S. Alam N. Gourinath S. Proteins. 2008; 72: 1222-1232Crossref PubMed Scopus (35) Google as this was in a different orientation compared with other OASS structures, and it a near the active But binding studies with the C-terminal peptides of SAT to EhOASS clearly shows that the loss of is due to differences in the C-terminal of EhSAT1 EhOASS is known from studies that EhSAT1 does not interact with EhOASS (13Nozaki T. Asai T. Kobayashi S. Ikegami F. Noji M. Saito K. Takeuchi T. Mol. Biochem. Parasitol. 1998; 97: 33-44Crossref PubMed Scopus (82) Google Scholar, T. Asai T. Sanchez L.B. Kobayashi S. Nakazawa M. Takeuchi T. J. Biol. Chem. 1999; 274: 32445-32452Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The inhibition and binding studies of EhOASS with peptide also that does bind to the EhOASS active site, confirming studies. But different SAT C-terminal inhibition of EhOASS and also high binding We to if the of EhSAT1 was mutated to this mutant bind to the EhOASS activity was inhibited by about in the of that if a different C-terminal sequence was in EhSAT1, it could have with EhOASS, and E. histolytica could have a complex regulatory cysteine E. histolytica is of and which are in mechanisms in Cysteine was shown to be the major thiol in defense of E. and it also plays a role in the survival of this to elongation, and growth (13Nozaki T. Asai T. Kobayashi S. Ikegami F. Noji M. Saito K. Takeuchi T. Mol. Biochem. Parasitol. 1998; 97: 33-44Crossref PubMed Scopus (82) Google Scholar). The EhSAT1 structure that E. histolytica has in a that the complex regulatory mechanisms are with a of the N-terminal and C-terminal and an in the loop near the active site that still maintained its enzyme The in the C-terminal region and in the loop near the active site to an acetyl-CoA binding site, as the C-terminal region in EhSAT1 does not this Cys binding does not inhibit the acetyl-CoA binding and with Ser for the active in the N-terminal region of EhSAT1 compared with that of other SATs to trimer formation and loss of hexamer This trimer in form a large cysteine synthase complex of SAT and two dimers of thus, to the loss of and this The pathway is regulated by feedback inhibition of thus, that the has Cys to from oxidative exists as a trimer both in solution and in crystal The differences in the N-terminal domain and its charge be responsible for not hexamers as in and C-terminal end and which are different from and do not interact with each other in the complex structure, the acetyl-CoA binding site These differences are responsible for loss of inhibition kinetics to acetyl-CoA by Cys. Ser and Cys bind to the same in the active site, with clearly that Cys inhibits SAT by competitive inhibition with Ser. In and the Cys inhibits both acetyl-CoA binding as well as serine whereas Cys inhibits Ser binding to EhSAT1, which is clearly by the complex is there at the C-terminal EhSAT1 bind with the EhOASS active the C-terminal of EhSAT1 had with is any of EhSAT1 could have with EhOASS and could have formed a complex. But this complex have been a rather than a as in other This loss of a complex regulatory mechanism here by structural features that hexamer by Cys not acetyl-CoA and by cysteine synthase complex ensure that there is Cys available in the E. histolytica for its survival. IntroductionIn bacteria and plants, l-cysteine is synthesized from l-Serine by two key enzymes serine acetyltransferase (SAT) 3The abbreviations used are: SAT, serine acetyltransferase; OASS, O-acetyl serine (OAS) sulfhydrylase; DTNB, 5,5′-dithio-bis(2-nitrobenzoic acid); PLP, pyridoxal-5′-phosphate; TNB, 5-thio(2-nitrobenzoate); Ec-, E. coli; Hi-, H. influenzae; Eh, E. histolytica; r.m.s.d., root mean square deviation. and O-acetyl serine sulfhydrylase (OASS). SAT converts l-Serine to O-acetyl serine by transferring an acetyl group from acetyl-CoA. Later, OASS converts the O-acetyl serine (OAS) to l-cysteine by adding a sulfide. This cysteine biosynthetic pathway contributes significantly to the incorporation of inorganic sulfur into organic compounds. SAT is in low abundance compared with OASS and is the rate-limiting component in this pathway (1Smith I.K. Thompson J.F. Biochim. Biophys. Acta. 1971; 227: 288-295Crossref PubMed Scopus (58) Google Scholar).The intracellular level of Cys is maintained by complex kinetics and strictly regulated by two known mechanisms. In the first, SAT is inhibited by Cys through a feedback mechanism. The second mechanism involves association of SAT and OASS to form a cysteine synthase complex. In the complex form, SAT activity increases, whereas OASS activity is reduced (2Saito K. Yokoyama H. Noji M. Murakoshi I. J. Biol. Chem. 1995; 270: 16321-16326Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This results in excess production of OAS, which causes the dissociation of the cysteine synthase complex and subsequent down-regulation of SAT activity (3Kredich N.M. Becker M.A. Tomkins G.M. J. Biol. Chem. 1969; 244: 2428-2439Abstract Full Text PDF PubMed Google Scholar).The structures of SAT from Escherichia coli (EcSAT) and Haemophilus influenzae (HiSAT) have been reported (4Pye V.E. Tingey A.P. Robson R.L. Moody P.C. J. Biol. Chem. 2004; 279: 40729-40736Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 5Gorman J. Shapiro L. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 1600-1605Crossref PubMed Scopus (29) Google Scholar) and also in complex with cysteine and acetyl-CoA (6Olsen L.R. Huang B. Vetting M.W. Roderick S.L. Biochemistry. 2004; 43: 6013-6019Crossref PubMed Scopus (67) Google Scholar). These structures exist as hexamers in the form of dimer of trimers. The solution studies and structures of OASS reveal that it stays as a dimer (7Krishna C. Jain R. Kashav T. Wadhwa D. Alam N. Gourinath S. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2007; 63: 512-515Crossref PubMed Scopus (10) Google Scholar, 8Becker M.A. Kredich N.M. Tomkins G.M. J. Biol. Chem. 1969; 244: 2418-2427Abstract Full Text PDF PubMed Google Scholar, 9Chinthalapudi K. Kumar M. Kumar S. Jain S. Alam N. Gourinath S. Proteins. 2008; 72: 1222-1232Crossref PubMed Scopus (35) Google Scholar). Biochemical and modeling studies indicated that one SAT hexamer and two OASS dimers interact to form the cysteine synthase complex (10Feldman-Salit A. Wirtz M. Hell R. Wade R.C. J. Mol. Biol. 2009; 386: 37-59Crossref PubMed Scopus (65) Google Scholar). The molecular details of the cysteine synthase complex are still not very clear. Various biochemical and structural studies have revealed that C-terminal end of SAT interacts with the active site of OASS (11Zhao C. Moriga Y. Feng B. Kumada Y. Imanaka H. Imamura K. Nakanishi K. Biochem. Biophys. Res. Commun. 2006; 341: 911-916Crossref PubMed Scopus (42) Google Scholar, 27Denk D. Böck A. J. Gen. Microbiol. 1987; 133: 515-525PubMed Google Scholar).Entamoeba histolytica is the enteric protozoan parasite that causes amoebic colitis and extra-intestinal abscesses in approximately 50 million people and kills about 70,000 each year (12WHO/PAHO/UNESCO Epidemiol. Bull. 1997; 18: 13-14Google Scholar). Cysteine was reported as the major thiol in E. histolytica (13Nozaki T. Asai T. Kobayashi S. Ikegami F. Noji M. Saito K. Takeuchi T. Mol. Biochem. Parasitol. 1998; 97: 33-44Crossref PubMed Scopus (82) Google Scholar) and is assumed to play a major role in oxidative stress defense mechanisms in this glutathione-deficient organism. Cysteine has also been shown to be necessary for survival, growth, elongation, and attachment to matrix (14Nozaki T. Asai T. Sanchez L.B. Kobayashi S. Nakazawa M. Takeuchi T. J. Biol. Chem. 1999; 274: 32445-32452Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 15Nozaki T. Ali V. Tokoro M. Adv. Parasitol. 2005; 60: 1-99Crossref PubMed Scopus (94) Google Scholar). The cysteine biosynthetic pathway could be crucial target to develop new chemotherapeutics (16Agarwal S.M. Jain R. Bhattacharya A. Azam A. Int. J. Parasitol. 2008; 38: 137-141Crossref PubMed Scopus (37) Google Scholar). Similar to other bacteria and plants, l-cysteine is synthesized by serine acetyltransferase (EhSAT) and O-acetyl serine sulfhydrylase (EhOASS) in E. histolytica. But unlike in other bacteria and plants, EhSAT and EhOASS do not interact with each other and do not form the cysteine synthase complex (14Nozaki T. Asai T. Sanchez L.B. Kobayashi S. Nakazawa M. Takeuchi T. J. Biol. Chem. 1999; 274: 32445-32452Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar).The fact that the E. histolytica genome encodes three forms of SAT suggests the importance of this enzyme for its survival. The EhSAT1 and EhSAT2 isoforms are very similar (78% identity), whereas the EhSAT3 isoform has very low sequence homology with EhSAT1 and EhSAT2 (48% identity). These three isoforms show differences in sensitivity to the feedback inhibitor l-cysteine (17Hussain S. Ali V. Jeelani G. Nozaki T. Mol. Biochem. Parasitol. 2009; 163: 39-47Crossref PubMed Scopus (32) Google Scholar). Till now there is no structural information available as to how sensitivity for feedback inhibition of cysteine is lost for some mutants/isoforms and why cysteine synthase complex does not form in E. histolytica.Here for the first time we report the structure of EhSAT at 1.77 Å resolution as well as its structure bound with its substrate serine at 1.59 Å resolution and its inhibitor cysteine at 1.78 Å resolution. EhSAT1 in these structures is trimeric rather than hexameric. The structures with Ser and Cys indicate how Cys can be a competitive inhibitor with Ser and does not inhibit acetyl-CoA. The docking and fluorescence binding studies with EhOASS clearly show that if the EhSAT1 had different C-terminal sequence, EhSAT1 and EhOASS could interact with each other. The C-terminal mutant of EhSAT1 does inhibit EhOASS, confirming our docking studies. These new structural features and biochemical studies suggest abolition of cysteine synthase complex formation to ensure ample supply of cysteine for the survival of the organism.