Mechanism of Chalcone Synthase
2000; Elsevier BV; Volume: 275; Issue: 50 Linguagem: Inglês
10.1074/jbc.m008569200
ISSN1083-351X
AutoresJoseph M. Jez, Joseph P. Noel,
Tópico(s)Synthesis and biological activity
ResumoPolyketide synthases (PKS) assemble structurally diverse natural products using a common mechanistic strategy that relies on a cysteine residue to anchor the polyketide during a series of decarboxylative condensation reactions that build the final reaction product. Crystallographic and functional studies of chalcone synthase (CHS), a plant-specific PKS, indicate that a cysteine-histidine pair (Cys164-His303) forms part of the catalytic machinery. Thiol-specific inactivation and the pH dependence of the malonyl-CoA decarboxylation reaction were used to evaluate the potential interaction between these two residues. Inactivation of CHS by iodoacetamide and iodoacetic acid targets Cys164 in a pH-dependent manner (pKa = 5.50). The acidic pKa of Cys164 suggests that an ionic interaction with His303 stabilizes the thiolate anion. Consistent with this assertion, substitution of a glutamine for His303 maintains catalytic activity but shifts the pKa of the thiol to 6.61. Although the H303A mutant was catalytically inactive, the pH-dependent incorporation of [14C]iodoacetamide into this mutant exhibits a pKa = 7.62. Subsequent analysis of the pH dependence of the malonyl-CoA decarboxylation reaction catalyzed by wild-type CHS and the H303Q and C164A mutants also supports the presence of an ion pair at the CHS active site. Structural and sequence conservation of a cysteine-histidine pair in the active sites of other PKS implies that a thiolate-imidazolium ion pair plays a central role in polyketide biosynthesis. Polyketide synthases (PKS) assemble structurally diverse natural products using a common mechanistic strategy that relies on a cysteine residue to anchor the polyketide during a series of decarboxylative condensation reactions that build the final reaction product. Crystallographic and functional studies of chalcone synthase (CHS), a plant-specific PKS, indicate that a cysteine-histidine pair (Cys164-His303) forms part of the catalytic machinery. Thiol-specific inactivation and the pH dependence of the malonyl-CoA decarboxylation reaction were used to evaluate the potential interaction between these two residues. Inactivation of CHS by iodoacetamide and iodoacetic acid targets Cys164 in a pH-dependent manner (pKa = 5.50). The acidic pKa of Cys164 suggests that an ionic interaction with His303 stabilizes the thiolate anion. Consistent with this assertion, substitution of a glutamine for His303 maintains catalytic activity but shifts the pKa of the thiol to 6.61. Although the H303A mutant was catalytically inactive, the pH-dependent incorporation of [14C]iodoacetamide into this mutant exhibits a pKa = 7.62. Subsequent analysis of the pH dependence of the malonyl-CoA decarboxylation reaction catalyzed by wild-type CHS and the H303Q and C164A mutants also supports the presence of an ion pair at the CHS active site. Structural and sequence conservation of a cysteine-histidine pair in the active sites of other PKS implies that a thiolate-imidazolium ion pair plays a central role in polyketide biosynthesis. polyketide synthase chalcone synthase (EC 2.3.1.74) coenzyme A fatty acid synthase (3-[1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid Polyketide synthases (PKS)1 from bacteria, fungi, and plants produce an array of natural products (1Hopwood D.A. Sherman D.H. Annu. Rev. Genet. 1990; 23: 37-66Crossref Google Scholar, 2Hopwood D.A. Chem. Rev. 1997; 97: 2365-2397Crossref Scopus (621) Google Scholar, 3Schröder J. Trends Plant Sci. 1997; 2: 373-378Abstract Full Text PDF Google Scholar, 4Khosla C. Gokhale R.S. Jacobsen J.R. Cane D.E. Annu. Rev. Biochem. 1999; 68: 219-253Crossref PubMed Scopus (296) Google Scholar). Many polyketides possess pharmacological properties and are used as antibiotics, immunosuppressants, anti-cancer agents, and anti-fungal agents (5Hutchinson C.R. Curr. Opin. Microbiol. 1998; 1: 319-329Crossref PubMed Scopus (112) Google Scholar, 6Bentley R. Bennett J.W. Annu. Rev. Microbiol. 1999; 53: 411-446Crossref PubMed Scopus (76) Google Scholar). Despite the structural complexity of these compounds, a common chemical strategy underlies the biosynthetic mechanisms of different PKS. The initial reaction step involves loading a starter molecule onto an active site cysteine through an acyltransferase activity. Following formation of the primed acyl-enzyme complex, a decarboxylative condensation reaction extends the reaction intermediate. The elongation step can be repeated until an appropriate chain length is reached and the reaction product released. This process is analogous to the reactions catalyzed by fatty acid synthases (FAS) (7Wakil R.J. Biochemistry. 1989; 28: 4523-4540Crossref PubMed Scopus (678) Google Scholar). Recent structural and kinetic studies of chalcone synthase (CHS), a plant-specific PKS, have elucidated the basis of polyketide formation in plants and provide a model for understanding the reaction mechanism of other PKS.Unlike the modular PKS, such as 6-deoxyerythronolide B synthase, which are large protein assemblies with distinct active sites that catalyze each elongation step (1Hopwood D.A. Sherman D.H. Annu. Rev. Genet. 1990; 23: 37-66Crossref Google Scholar, 2Hopwood D.A. Chem. Rev. 1997; 97: 2365-2397Crossref Scopus (621) Google Scholar, 4Khosla C. Gokhale R.S. Jacobsen J.R. Cane D.E. Annu. Rev. Biochem. 1999; 68: 219-253Crossref PubMed Scopus (296) Google Scholar, 6Bentley R. Bennett J.W. Annu. Rev. Microbiol. 1999; 53: 411-446Crossref PubMed Scopus (76) Google Scholar), the plant-specific PKS function as homodimeric iterative PKS (monomer, molecular mass ∼ 42 kDa) that perform consecutive elongation reactions at two independent active sites (3Schröder J. Trends Plant Sci. 1997; 2: 373-378Abstract Full Text PDF Google Scholar, 8Tropf S. Kärcher B. Schröder G. Schröder J. J. Biol. Chem. 1995; 270: 7922-7928Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). CHS uses p-coumaroyl-CoA as a starter molecule and three malonyl-CoA extender molecules to form a tetraketide intermediate that is cyclized into 4,2′,4′,6′-tetrahydroxychalcone (chalcone) (Fig. 1 a) (9Kreuzaler F. Hahlbrock K. Eur. J. Biochem. 1975; 56: 203-223Crossref Scopus (150) Google Scholar). This activity is central to the biosynthesis of anti-microbial isoflavonoid phytoalexins, anthocyanin floral pigments, and flavonoid inducers ofRhizobium nodulation genes (10Dixon R.A. Paiva N.L. Plant Cell. 1995; 7: 1085-1097Crossref PubMed Scopus (3548) Google Scholar, 11Long S.R. Cell. 1989; 56: 203-214Abstract Full Text PDF PubMed Scopus (489) Google Scholar). Also, flavonoids are of interest as pharmacological agents (12Edwards M.L. Stemerick D.M. Sunkara P.S. J. Med. Chem. 1990; 33: 1948-1954Crossref PubMed Scopus (271) Google Scholar, 13Li R. Kenyon G.L. Cohen F.E. Chen X. Gong B. Dominguez J.N. Davidson E. Kurzban G. Miller R.E. Nuzum E.O. Rosenthal P.J. McKerrow J.H. J. Med. Chem. 1995; 38: 5031-5037Crossref PubMed Scopus (413) Google Scholar, 14Zwaagstra M.E. Timmerman H. Tamura M. Tohma T. Wada Y. Onogi K. Zhang M.-Q. J. Med. Chem. 1997; 40: 1075-1089Crossref PubMed Scopus (55) Google Scholar) and are constituents in plant-rich diets associated with a reduced risk of cardiovascular disease and some forms of cancer (15Birt D.F. Pelling J.C. Nair S. Lepley D. Prog. Clin. Biol. Res. 1993; 395: 223-234Google Scholar, 16Setchell K.D. Cassidy A. J. Nutr. 1993; 129: 758S-767SCrossref Google Scholar).The three-dimensional structure of alfalfa CHS2 provides a view of the active site that catalyzes chalcone formation (Fig. 1 b) (17Ferrer J.-L. Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Nat. Struct. Biol. 1999; 6: 775-784Crossref PubMed Scopus (559) Google Scholar). Four residues (Cys164, His303, Asn336, and Phe215) form the catalytic center of CHS and are strictly conserved in other CHS-like enzymes, including 2-pyrone synthase (18Helaruitta Y. Kotilainen M. Elomaa P. Kalkkinen N. Bremer K. Teeri T.H. Albert V.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9033-9038Crossref PubMed Scopus (78) Google Scholar), stilbene synthase (19Schröder G. Brown J.W.S. Schröder J. Eur. J. Biochem. 1988; 172: 161-169Crossref PubMed Scopus (191) Google Scholar), bibenzyl synthase (20Preisig-Müller R. Gnau P. Kindl H. Arch. Biochem. Biophys. 1995; 317: 201-207Crossref PubMed Scopus (57) Google Scholar), acridone synthase (21Junghanns K.T. Kneusel R.E. Baumert A. Maier W. Groger D. Matern U. Plant Mol. Biol. 1995; 27: 681-692Crossref PubMed Scopus (57) Google Scholar), and the rppA CHS-like protein (22Funa N. Ohnishi Y. Fujii I. Shibuya M. Ebizuka Y. Horinouchi S. Nature. 1999; 400: 897-899Crossref PubMed Scopus (234) Google Scholar). Based upon structural and functional studies of CHS (17Ferrer J.-L. Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Nat. Struct. Biol. 1999; 6: 775-784Crossref PubMed Scopus (559) Google Scholar, 23Lanz T Tropf S. Marner F.-J. Schröder G. Schröder J. J. Biol. Chem. 1991; 266: 9971-9976Abstract Full Text PDF PubMed Google Scholar, 24Jez J.M. Ferrer J.-L. Bowman M.E. Dixon R.A. Noel J.P. Biochemistry. 2000; 39: 890-902Crossref PubMed Scopus (283) Google Scholar), the proposed reaction mechanism involves Cys164 acting as the nucleophilic thiolate in the loading reaction and as the covalent thioester-anchor for the acyl-enzyme chain during the elongation reactions (Fig. 1 c). In addition, His303 and Asn336 catalyze the decarboxylation of malonyl-CoA in the elongation reaction and stabilize the transition state during the condensation phases of polyketide formation. Phe215 may orient substrates and reaction intermediates at the active site.In the crystal structure of CHS (17Ferrer J.-L. Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Nat. Struct. Biol. 1999; 6: 775-784Crossref PubMed Scopus (559) Google Scholar), the Sγ of Cys164forms a hydrogen bond with the Nε of His303, which is 3.5 Å distant (Fig. 1 b). Previous mutagenesis studies suggest that His303 does not act as a general base by abstracting a proton from Cys164 to form the reactive thiolate required for chalcone formation (24Jez J.M. Ferrer J.-L. Bowman M.E. Dixon R.A. Noel J.P. Biochemistry. 2000; 39: 890-902Crossref PubMed Scopus (283) Google Scholar). Rather, at physiological pH, these two residues may form a stable imidazolium-thiolate ion pair. In other enzymes, notably the cysteine proteases (25Lewis S.D. Johnson F.A. Shafer J.A. Biochemistry. 1976; 15: 5009-5017Crossref PubMed Scopus (129) Google Scholar, 26Lewis S.D. Johnson F.A. Shafer J.A. Biochemistry. 1981; 20: 48-51Crossref PubMed Scopus (153) Google Scholar, 27Polgar L. Csoma C. J. Biol. Chem. 1987; 262: 14448-14453Abstract Full Text PDF PubMed Google Scholar, 28Storer A.C. Menard R. Methods Enzymol. 1994; 234: 486-500Crossref Scopus (225) Google Scholar), the thiolate anion of the catalytic cysteine is stabilized by an imidazolium ion on an adjacent histidine. The structural proximity of Cys164 and His303 in the CHS active site raises the potential for a similar mechanistic feature in this PKS.This paper describes the use of thiol-specific inactivators to evaluate the reactivity of Cys164, to determine the pKa of the active site cysteine, and to establish the role of His303 in maintaining the reactivity of Cys164. In addition, the pH dependence of the malonyl-CoA decarboxylation reactions catalyzed by wild-type CHS and the H303Q and C164A mutants was examined. These studies demonstrate that Cys164 is a reactive thiolate anion with an acidic pKa that is modulated by interaction with His303. Combined with previous crystallographic and kinetic studies, this work provides insight on the mechanism of CHS and suggests that a thiolate-imidazolium ion pair plays a significant role in both polyketide and fatty acid biosynthesis.DISCUSSIONIodoacetamide and iodoacetic acid inactivate CHS through specific modification of Cys164 at the active site. This result agrees with mutagenesis experiments of CHS in which substitution of the cysteine with a serine or alanine results in a complete loss of chalcone formation (23Lanz T Tropf S. Marner F.-J. Schröder G. Schröder J. J. Biol. Chem. 1991; 266: 9971-9976Abstract Full Text PDF PubMed Google Scholar, 24Jez J.M. Ferrer J.-L. Bowman M.E. Dixon R.A. Noel J.P. Biochemistry. 2000; 39: 890-902Crossref PubMed Scopus (283) Google Scholar). Importantly, the pKa of Cys164 indicates that a thiolate anion is present at the CHS active site at physiological pH to serve as the nucleophile in the loading reaction and as the attachment site of the polyketide during the elongation reactions. The acidic pKa of Cys164 also explains why the sulfhydryl group of this residue is oxidized to sulfinic acid (Cys-SO2H) in several crystal structures of CHS (17Ferrer J.-L. Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Nat. Struct. Biol. 1999; 6: 775-784Crossref PubMed Scopus (559) Google Scholar, 24Jez J.M. Ferrer J.-L. Bowman M.E. Dixon R.A. Noel J.P. Biochemistry. 2000; 39: 890-902Crossref PubMed Scopus (283) Google Scholar). Similar oxidation of the active site thiol in the cysteine proteases also occurs (37Kamphuis I.G. Kalk K.H. Swarte M.B.A. Drenth J. J. Mol. Biol. 1984; 179: 233-256Crossref PubMed Scopus (461) Google Scholar, 38Kamphuis I.G. Drenth J. Baker E.N. J. Mol. Biol. 1985; 182: 317-329Crossref PubMed Scopus (244) Google Scholar). In these enzymes, the reactive thiolate forms an ion pair with the imidazolium ion of an adjacent histidine residue (27Polgar L. Csoma C. J. Biol. Chem. 1987; 262: 14448-14453Abstract Full Text PDF PubMed Google Scholar). Although the pKa value determined for Cys164 in CHS is not as low as the 3.3–4.0 pKa of the thiolate present at the active site of the cysteine proteases (25Lewis S.D. Johnson F.A. Shafer J.A. Biochemistry. 1976; 15: 5009-5017Crossref PubMed Scopus (129) Google Scholar, 26Lewis S.D. Johnson F.A. Shafer J.A. Biochemistry. 1981; 20: 48-51Crossref PubMed Scopus (153) Google Scholar, 27Polgar L. Csoma C. J. Biol. Chem. 1987; 262: 14448-14453Abstract Full Text PDF PubMed Google Scholar), the observed pKa of the CHS active site thiol is significantly shifted from the accepted pKa value of 8.0–8.5 for a cysteine sulfhydryl moiety (35Walsh C.T. Enzymatic Reaction Mechanisms. W. H. Freeman, New York1979: 26Google Scholar). A reduction in pKa of this magnitude requires stabilization by the local environment. The proximity of His303 to Cys164 in the CHS structure suggests that the histidine, as an imidazolium cation, stabilizes the thiolate anion (Fig. 8).Figure 8Proposed interactions at the active site of wild-type and mutant CHS. The catalytic efficiencies of each enzyme (24Jez J.M. Ferrer J.-L. Bowman M.E. Dixon R.A. Noel J.P. Biochemistry. 2000; 39: 890-902Crossref PubMed Scopus (283) Google Scholar) and the pKa of inactivation are summarized.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Consistent with the presence of an ion pair at the CHS active site, the reactivity and pKa of Cys164 shifted when mutations of His303 were made. Substitution of a glutamine for His303 also maintains the thiolate of Cys164, but the resulting nucleophile is less reactive than in wild-type CHS. In the three-dimensional structure of the H303Q mutant (24Jez J.M. Ferrer J.-L. Bowman M.E. Dixon R.A. Noel J.P. Biochemistry. 2000; 39: 890-902Crossref PubMed Scopus (283) Google Scholar), substitution of a glutamine for His303 is isosteric with the amide nitrogen of Gln303 hydrogen bonding the sulfhydryl of Cys164. Hydrogen bonding and a partial positive charge on the amide nitrogen arising from resonance stabilization promote formation of the thiolate at Cys164, albeit less efficiently and with a corresponding shift in pKa (Fig. 8).No stabilizing effect would be expected from the side chain of Ala303 in the H303A mutant (Fig. 8). As described previously (24Jez J.M. Ferrer J.-L. Bowman M.E. Dixon R.A. Noel J.P. Biochemistry. 2000; 39: 890-902Crossref PubMed Scopus (283) Google Scholar), the lack of malonyl-CoA decarboxylation and chalcone formation activities in this mutant underscores the mechanistic importance of the histidine. Although the pKa for iodoacetamide labeling of the H303A mutant is shifted to 7.62, the observed pKa is still 0.4–0.9 pH units below that of a free cysteine. Asn336 is another polar residue near Cys164 (4.1 Å), but substitution of the asparagine with an alanine does not alter the inactivation kinetics of the N336A mutant or the pKa of Cys164 in this mutant. 2J. M. Jez and J. P. Noel, unpublished observations. Since no other direct interactions occur with Cys164, the local environment may alter the pKa of this residue. Cys164 is at the N terminus of α-helix 9 in the CHS structure and the helix-dipole may further reduce the thiol's pKa in the absence of other interactions (40Kortemme T. Creighton T.E. J. Mol. Biol. 1995; 253: 799-812Crossref PubMed Scopus (280) Google Scholar, 41Dillet V. Dyson H.J. Bashford D. Biochemistry. 1998; 37: 10298-10396Crossref PubMed Scopus (78) Google Scholar, 42Davies C. Heath R.J. White S.W. Rock C.O. Structure. 2000; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar).If Cys164 and His303 form a stable ion pair, then the ionization states of both residues should be thermodynamically linked and a second inflection point observed in the pH profiles for inactivation. For example, in papain the pKa of the active site histidine shifts from 4.3 to 8.5 when the active site thiol is deprotonated (25Lewis S.D. Johnson F.A. Shafer J.A. Biochemistry. 1976; 15: 5009-5017Crossref PubMed Scopus (129) Google Scholar, 26Lewis S.D. Johnson F.A. Shafer J.A. Biochemistry. 1981; 20: 48-51Crossref PubMed Scopus (153) Google Scholar). Since the structure of wild-type CHS indicates that interaction between Cys164 and His303 occurs (17Ferrer J.-L. Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Nat. Struct. Biol. 1999; 6: 775-784Crossref PubMed Scopus (559) Google Scholar, 24Jez J.M. Ferrer J.-L. Bowman M.E. Dixon R.A. Noel J.P. Biochemistry. 2000; 39: 890-902Crossref PubMed Scopus (283) Google Scholar), it is likely that the absent inflection point is beyond the pH range studied. Therefore, the potential pKa of His303 in the free enzyme must be greater than 9.0, which is also consistent with the activity of the H303Q mutant, in which the glutamine's amide pKa is approximately 17.0 (39Kyte J. Structure in Protein Chemistry. Garland Publishing, New York1995: 59Google Scholar). Under physiological conditions, a stable thiolate-imidazolium ion pair in the CHS active site would maintain the nucleophilic thiolate required for the loading reaction.The pH dependence of the malonyl-CoA decarboxylation reaction also supports the importance of a charged interaction at the CHS active site. In wild-type enzyme, the presence of an imidazolium ion would enhance formation of an enolate anion in the decarboxylation reaction (43O'Leary M.H. Sigman D.S. Boyer P.D. The Enzymes. Academic Press, New York1992: 235-269Google Scholar). Since the kcat pH profile corresponds to the protonation state of the enzyme-substrate complex, the assignment of the observed pKa to any particular residue is problematic. Most likely, the observed break point represents enolization of malonyl-CoA during the decarboxylation reaction. Mutation of either His303 or Cys164 to an aprotic side chain eliminates the observed inflection point, suggesting that the rate-determining step in the decarboxylation reaction is altered. Although the H303Q and C164A mutants retain hydrogen bond donors at the active site as a glutamine and a histidine, respectively, the ionic pair at the active site is disrupted. Loss of the imidazolium ion in these mutants would slow substrate enolization and eliminate the observed pH dependence on the decarboxylation reaction.Since the active site residues of CHS are conserved among CHS-like PKS, including 2-pyrone synthase (18Helaruitta Y. Kotilainen M. Elomaa P. Kalkkinen N. Bremer K. Teeri T.H. Albert V.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9033-9038Crossref PubMed Scopus (78) Google Scholar), stilbene synthase (19Schröder G. Brown J.W.S. Schröder J. Eur. J. Biochem. 1988; 172: 161-169Crossref PubMed Scopus (191) Google Scholar), bibenzyl synthase (20Preisig-Müller R. Gnau P. Kindl H. Arch. Biochem. Biophys. 1995; 317: 201-207Crossref PubMed Scopus (57) Google Scholar), acridone synthase (21Junghanns K.T. Kneusel R.E. Baumert A. Maier W. Groger D. Matern U. Plant Mol. Biol. 1995; 27: 681-692Crossref PubMed Scopus (57) Google Scholar), and the rppA CHS-like protein (22Funa N. Ohnishi Y. Fujii I. Shibuya M. Ebizuka Y. Horinouchi S. Nature. 1999; 400: 897-899Crossref PubMed Scopus (234) Google Scholar), these enzymes likely retain a thiolate-imidazolium ion pair at their active sites. In addition, homology of the CHS active site residues with those of the FAS and other PKS (44Siggaard-Andersen M. Protein Sequences Data Anal. 1993; 5: 325-335Google Scholar), such as 6-deoxyerythronolide B synthase and actinorhodin synthase, suggests that a similar ion pair may be a defining feature in these enzymes.Functional studies of FAS II and III demonstrate that the role of the catalytic cysteine in these enzymes is identical to that of Cys164 in CHS (45Witkowski A. Joshi A.K. Lindqvist Y. Smith S. Biochemistry. 1999; 38: 11643-11650Crossref PubMed Scopus (73) Google Scholar, 46Abbadi A. Brummel M. Schutt B.S. Slabaugh M.B. Schuch R. Spener F. Biochem. J. 2000; 345: 153-160Crossref PubMed Google Scholar). Also, the rapid inactivation of FAS by iodoacetamide implies that the active site thiol is highly reactive (47Oesterhelt D. Bauer H. Kresze G.-B. Steber L. Lynen F. Eur. J. Biochem. 1977; 79: 173-180Crossref PubMed Scopus (43) Google Scholar, 48Stoops J.K. Henry S.J. Wakil S.J. J. Biol. Chem. 1983; 258: 12482-12486Abstract Full Text PDF PubMed Google Scholar). Although studies on FAS III demonstrate the importance of the active site histidine in the overall reaction mechanism (42Davies C. Heath R.J. White S.W. Rock C.O. Structure. 2000; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 46Abbadi A. Brummel M. Schutt B.S. Slabaugh M.B. Schuch R. Spener F. Biochem. J. 2000; 345: 153-160Crossref PubMed Google Scholar), the effect of this residue on the pKa of the catalytic cysteine in FAS has not been evaluated. Currently, no detailed structural information is available on the modular or heterodimeric iterative PKS, but mechanistic analysis of actinorhodin synthase suggests that a cysteine-histidine dyad is an essential catalytic component (49Dreier J. Khosla C. Biochemistry. 2000; 39: 2088-2095Crossref PubMed Scopus (64) Google Scholar). In the reaction mechanism of actinorhodin synthase, a cysteine (Cys169) serves as the attachment point for the polyketide chain (49Dreier J. Khosla C. Biochemistry. 2000; 39: 2088-2095Crossref PubMed Scopus (64) Google Scholar, 50Kim E.-S. Cramer K.D. Shreve A.L. Sherman D.H. J. Bacteriol. 1995; 177: 1202-1207Crossref PubMed Google Scholar, 51He M. Varoglu M. Sherman D.H. J. Bacteriol. 2000; 182: 2619-2623Crossref PubMed Scopus (24) Google Scholar), and a histidine (His346) may activate the thiol in the loading and elongation reactions.Stabilization of negatively charged thiolates by imidazolium ions at the active sites of PKS eliminates the need for formal proton transfers such as those governed by general acid-base catalysis. Intuitively, the catalytic advantage of this mechanistic model may derive from limiting bond making and bond breaking steps to thioester formation and breakdown without additional proton transfers to and from the thiolate and imidazolium ions and their respective acids and bases. Polyketide synthases (PKS)1 from bacteria, fungi, and plants produce an array of natural products (1Hopwood D.A. Sherman D.H. Annu. Rev. Genet. 1990; 23: 37-66Crossref Google Scholar, 2Hopwood D.A. Chem. Rev. 1997; 97: 2365-2397Crossref Scopus (621) Google Scholar, 3Schröder J. Trends Plant Sci. 1997; 2: 373-378Abstract Full Text PDF Google Scholar, 4Khosla C. Gokhale R.S. Jacobsen J.R. Cane D.E. Annu. Rev. Biochem. 1999; 68: 219-253Crossref PubMed Scopus (296) Google Scholar). Many polyketides possess pharmacological properties and are used as antibiotics, immunosuppressants, anti-cancer agents, and anti-fungal agents (5Hutchinson C.R. Curr. Opin. Microbiol. 1998; 1: 319-329Crossref PubMed Scopus (112) Google Scholar, 6Bentley R. Bennett J.W. Annu. Rev. Microbiol. 1999; 53: 411-446Crossref PubMed Scopus (76) Google Scholar). Despite the structural complexity of these compounds, a common chemical strategy underlies the biosynthetic mechanisms of different PKS. The initial reaction step involves loading a starter molecule onto an active site cysteine through an acyltransferase activity. Following formation of the primed acyl-enzyme complex, a decarboxylative condensation reaction extends the reaction intermediate. The elongation step can be repeated until an appropriate chain length is reached and the reaction product released. This process is analogous to the reactions catalyzed by fatty acid synthases (FAS) (7Wakil R.J. Biochemistry. 1989; 28: 4523-4540Crossref PubMed Scopus (678) Google Scholar). Recent structural and kinetic studies of chalcone synthase (CHS), a plant-specific PKS, have elucidated the basis of polyketide formation in plants and provide a model for understanding the reaction mechanism of other PKS. Unlike the modular PKS, such as 6-deoxyerythronolide B synthase, which are large protein assemblies with distinct active sites that catalyze each elongation step (1Hopwood D.A. Sherman D.H. Annu. Rev. Genet. 1990; 23: 37-66Crossref Google Scholar, 2Hopwood D.A. Chem. Rev. 1997; 97: 2365-2397Crossref Scopus (621) Google Scholar, 4Khosla C. Gokhale R.S. Jacobsen J.R. Cane D.E. Annu. Rev. Biochem. 1999; 68: 219-253Crossref PubMed Scopus (296) Google Scholar, 6Bentley R. Bennett J.W. Annu. Rev. Microbiol. 1999; 53: 411-446Crossref PubMed Scopus (76) Google Scholar), the plant-specific PKS function as homodimeric iterative PKS (monomer, molecular mass ∼ 42 kDa) that perform consecutive elongation reactions at two independent active sites (3Schröder J. Trends Plant Sci. 1997; 2: 373-378Abstract Full Text PDF Google Scholar, 8Tropf S. Kärcher B. Schröder G. Schröder J. J. Biol. Chem. 1995; 270: 7922-7928Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). CHS uses p-coumaroyl-CoA as a starter molecule and three malonyl-CoA extender molecules to form a tetraketide intermediate that is cyclized into 4,2′,4′,6′-tetrahydroxychalcone (chalcone) (Fig. 1 a) (9Kreuzaler F. Hahlbrock K. Eur. J. Biochem. 1975; 56: 203-223Crossref Scopus (150) Google Scholar). This activity is central to the biosynthesis of anti-microbial isoflavonoid phytoalexins, anthocyanin floral pigments, and flavonoid inducers ofRhizobium nodulation genes (10Dixon R.A. Paiva N.L. Plant Cell. 1995; 7: 1085-1097Crossref PubMed Scopus (3548) Google Scholar, 11Long S.R. Cell. 1989; 56: 203-214Abstract Full Text PDF PubMed Scopus (489) Google Scholar). Also, flavonoids are of interest as pharmacological agents (12Edwards M.L. Stemerick D.M. Sunkara P.S. J. Med. Chem. 1990; 33: 1948-1954Crossref PubMed Scopus (271) Google Scholar, 13Li R. Kenyon G.L. Cohen F.E. Chen X. Gong B. Dominguez J.N. Davidson E. Kurzban G. Miller R.E. Nuzum E.O. Rosenthal P.J. McKerrow J.H. J. Med. Chem. 1995; 38: 5031-5037Crossref PubMed Scopus (413) Google Scholar, 14Zwaagstra M.E. Timmerman H. Tamura M. Tohma T. Wada Y. Onogi K. Zhang M.-Q. J. Med. Chem. 1997; 40: 1075-1089Crossref PubMed Scopus (55) Google Scholar) and are constituents in plant-rich diets associated with a reduced risk of cardiovascular disease and some forms of cancer (15Birt D.F. Pelling J.C. Nair S. Lepley D. Prog. Clin. Biol. Res. 1993; 395: 223-234Google Scholar, 16Setchell K.D. Cassidy A. J. Nutr. 1993; 129: 758S-767SCrossref Google Scholar). The three-dimensional structure of alfalfa CHS2 provides a view of the active site that catalyzes chalcone formation (Fig. 1 b) (17Ferrer J.-L. Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Nat. Struct. Biol. 1999; 6: 775-784Crossref PubMed Scopus (559) Google Scholar). Four residues (Cys164, His303, Asn336, and Phe215) form the catalytic center of CHS and are strictly conserved in other CHS-like enzymes, including 2-pyrone synthase (18Helaruitta Y. Kotilainen M. Elomaa P. Kalkkinen N. Bremer K. Teeri T.H. Albert V.A. Proc. Natl. Acad. Sci. U. S. 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