Mechanism of Chorismate Synthase

Chorismate synthase catalyzes the last step in the common shikimate pathway leading to aromatic compounds such as the aromatic amino acids. The reaction consists of the 1,4-anti-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate. Although this reaction does not involve a net redox change, the enzyme has an absolute requirement for reduced flavin mononucleotide, which is not consumed during the reaction. Two invariant histidine residues are found in the active site of the enzyme: His17 and His106. Using site-directed mutagenesis, both histidines were replaced by alanine, reducing the activity 10- and 20-fold in the H106A and H17A mutant protein, respectively. Based on the characterization of the two single mutant proteins, it is proposed that His106 serves to protonate the monoanionic reduced FMN, whereas His17 protonates the leaving phosphate group of the substrate. An enzymatic reaction mechanism in keeping with the experimental results is presented. Chorismate synthase catalyzes the last step in the common shikimate pathway leading to aromatic compounds such as the aromatic amino acids. The reaction consists of the 1,4-anti-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate. Although this reaction does not involve a net redox change, the enzyme has an absolute requirement for reduced flavin mononucleotide, which is not consumed during the reaction. Two invariant histidine residues are found in the active site of the enzyme: His17 and His106. Using site-directed mutagenesis, both histidines were replaced by alanine, reducing the activity 10- and 20-fold in the H106A and H17A mutant protein, respectively. Based on the characterization of the two single mutant proteins, it is proposed that His106 serves to protonate the monoanionic reduced FMN, whereas His17 protonates the leaving phosphate group of the substrate. An enzymatic reaction mechanism in keeping with the experimental results is presented. Chorismate synthase catalyzes the last step in the shikimate pathway leading to the branch point metabolite chorismate, which is utilized in a number of enzymatic transformations toward the biosynthesis of aromatic compounds such as the aromatic amino acids tyrosine, phenylalanine, and tryptophan (for a recent review, see Ref. 1Knaggs A.R. Nat. Prod. Rep. 2001; 18: 334-355Crossref PubMed Scopus (59) Google Scholar). This reaction involves an 1,4-anti-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate-3-phosphate (EPSP) 1The abbreviations used are: EPSP, 5-enolpyruvylshikimate 3-phosphate; DEPC, diethyl pyrocarbonate; MOPS, 3-(N-morpholino)propanesulfonic acid; NcCS, N. crassa chorismate synthase; HOMO, highest occupied molecular orbital.1The abbreviations used are: EPSP, 5-enolpyruvylshikimate 3-phosphate; DEPC, diethyl pyrocarbonate; MOPS, 3-(N-morpholino)propanesulfonic acid; NcCS, N. crassa chorismate synthase; HOMO, highest occupied molecular orbital. (2Floss H.G. Onderka D.K. Carroll M. J. Biol. Chem. 1972; 247: 736-744Abstract Full Text PDF PubMed Google Scholar, 3Morell H. Clark M.J. Knowles P.F. Sprinson D.B. J. Biol. Chem. 1967; 242: 82-90Abstract Full Text PDF PubMed Google Scholar). Although the overall redox state is not changed, the enzyme has an absolute requirement for a reduced FMN cofactor, as shown in Scheme 1. This latter finding gave rise to intense mechanistic studies on the putative functional role of reduced FMN in catalysis (reviewed in Refs. 4Bornemann S. Lowe D.J. Thorneley R.N.F. Biochem. Soc. Trans. 1996; 24: 84-88Crossref PubMed Scopus (14) Google Scholar and 5Macheroux P. Schmid J. Amrhein N. Schaller A. Planta. 1999; 207: 325-334Crossref PubMed Scopus (82) Google Scholar). From these studies, it has become evident that reduced FMN plays a pivotal role in the chorismate synthase catalyzed reaction (6Bornemann S. Lowe D.J. Thorneley R.N.F. Biochemistry. 1996; 35: 9907-9916Crossref PubMed Scopus (35) Google Scholar, 7Macheroux P. Bornemann S. Ghisla S. Thorneley R.N.F. J. Biol. Chem. 1996; 271: 25850-25858Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 8Macheroux P. Petersen J. Bornemann S. Lowe D.J. Thorneley R.N.F. Biochemistry. 1996; 35: 1643-1652Crossref PubMed Scopus (38) Google Scholar, 9Osborne A. Thorneley R.N.F. Abell C. Bornemann S. J. Biol. Chem. 2000; 275: 35825-35830Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). This conclusion has received further support by the first high resolution x-ray structure of chorismate synthase from the Gram-positive bacteria Streptococcus pneumoniae (10Maclean J. Ali S. Structure. 2003; 11: 1499-1511Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In the reported structure of the ternary complex, comprising FMN and EPSP as ligands, EPSP is stacked above the si-face of the isoalloxazine ring in an average distance of 3.3 Å. This close juxtaposition of FMN and EPSP supports earlier suggestions for a direct role of reduced FMN in the elimination reaction, such as a radical mechanism, in which electron transfer from the reduced cofactor to the substrate initiates C-O bond breakage of the phosphate group to yield a substrate-derived neutral radical (6Bornemann S. Lowe D.J. Thorneley R.N.F. Biochemistry. 1996; 35: 9907-9916Crossref PubMed Scopus (35) Google Scholar, 7Macheroux P. Bornemann S. Ghisla S. Thorneley R.N.F. J. Biol. Chem. 1996; 271: 25850-25858Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 8Macheroux P. Petersen J. Bornemann S. Lowe D.J. Thorneley R.N.F. Biochemistry. 1996; 35: 1643-1652Crossref PubMed Scopus (38) Google Scholar, 9Osborne A. Thorneley R.N.F. Abell C. Bornemann S. J. Biol. Chem. 2000; 275: 35825-35830Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 11Macheroux P. Bornemann S. Thorneley R.N.F. Stevenson K.J. Massey V. Williams C.H.J. Flavins and Flavoproteins 1996. University of Calgary Press, Calgary, Canada1997: 113-122Google Scholar). Since no net redox change occurs in the reaction, a reverse transfer of the transiently donated electron is required to complete the catalytic cycle. In this mechanism, which is unique among flavin-dependent enzymes, the flavin environment in the active site is thought to play a key role in tuning the flavin cofactor to such a radical transfer mechanism. Two classes of chorismate synthases are distinguished by their ability to use NADPH for the reduction of oxidized FMN. Those enzymes possessing this additional catalytic activity are called “bifunctional,” whereas those lacking it are called “monofunctional.” Bifunctional enzymes have only been found in yeasts (Neurospora crassa and Saccharomyces cerevisiae), with all other species apparently harboring monofunctional chorismate synthases. In contrast to the mode of flavin reduction, the two classes of chorismate synthases show no apparent difference in the mechanism of the elimination reaction. Multiple sequence alignments of chorismate synthases from bacterial, fungal, plant, and protozoan origin have revealed a number of invariant amino acid residues in monofunctional as well as bifunctional chorismate synthases, suggesting a pertinent role in catalysis in both classes of enzymes (12Macheroux P. Schönbrunn E. Svergun D.I. Volkov V.V. Koch M.H.J. Bornemann S. Thorneley R.N.F. Biochem. J. 1998; 335: 319-327Crossref PubMed Scopus (31) Google Scholar). Among these are two histidine residues, His17 and His106, in the bifunctional N. crassa enzyme, which are both found in the active site of chorismate synthase within 2.7 and 3 Å of the C(2) = O position of the flavin ring (His106) and the substrate phosphate group (His17), respectively, as shown in Scheme 2. The structural proximity of the imidazole ring of His17 to the substrate phosphate group as well as its suitable properties as an acid-base catalyst suggest that this residue protonates the leaving group upon C-O bond cleavage. The role of His106 is less clear; however, recent studies have provided evidence that the monoanionic form of reduced FMN (pKa = 6.7 free in solution (13Dudley K.H. Ehrenberg A. Hemmerich P. Müller F. Helv. Chim. Acta. 1964; 47: 1354-1383Crossref Scopus (153) Google Scholar)) is protonated upon binding of EPSP to the active site. Thus, His106 may be the general acid that protonates the N(1)-C(2) = O locus of the isoalloxazine moiety. The rationale behind the protonation of the reduced flavin is the assumption that a neutral reduced flavin has a more negative redox and may the electron transfer to the substrate more of the active site is the of an amino acid in the of the C-(6proR) the flavin is close to this that the is in bond the role of His17 and His106 in the catalysis of chorismate have two single histidine to mutant and by site-directed the of these amino acid on binding of oxidized and reduced FMN, binding of the substrate EPSP, the of a catalytic and enzymatic The results of that both residues are for catalytic activity and the of the flavin Based on a mechanism in which both residues as general acid were of the highest and from from and phosphate from and enzymes were from were from EPSP from 5-enolpyruvylshikimate 3-phosphate synthase and by high molecular were J. Scholar). of crassa Chorismate histidine to mutant were as for enzyme P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). enzyme as P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). from EPSP as a of enzyme activity a as by Ref. Biochem. PubMed Scopus Google acid were the site-directed from The as the The the were used for the is and reverse and reverse were the The were by were with a were with a The for tryptophan and for flavin it were in MOPS, reaction were a with a and a enzyme and substrate were by the with by of and of the the A. redox of oxidized FMN in the of and the two histidine mutant by the in Ref. V. S. Flavins and of the experimental are in Ref. 8Macheroux P. Petersen J. Bornemann S. Lowe D.J. Thorneley R.N.F. Biochemistry. 1996; 35: 1643-1652Crossref PubMed Scopus (38) Google = used as a of reduced FMN in its protonated and form were the from of by imidazole ring of histidine residues with DEPC, its as an acid-base catalyst 47: PubMed Scopus Google Scholar). of with in a not and of enzyme activity The enzyme activity of by a of of by of revealed that histidine residues were with not Among the histidine residues were the two invariant histidine residues His17 and His106 to the N. crassa chorismate synthase Although it is not to the of the enzyme to of the invariant the that both of these histidines are in the active site and that the enzyme gave a first that the two invariant histidines play an role in and of an H17A and H106A invariant histidine residues and were replaced by residues site-directed The two single histidine mutant and were and from in a as for enzyme P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). mutant and were by the for P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). and of the H17A and H106A mutant were with of the H17A and H106A histidine mutant were first for activity in a enzyme In this the activity of the H17A and H106A mutant and respectively, as with Since the is on the activity of which may be by the the activity in an FMN reduced to with chorismate synthase activity be from the The of chorismate in a these the H17A and H106A mutant and of the enzyme respectively. The of both mutant in the of that the activity is by the histidine to C-O bond breakage is by the amino acid a that the of phosphate from EPSP the activity in the H106A H17A with the the two histidine mutant phosphate occurs during catalysis and C-(6proR) hydrogen (6Bornemann S. Lowe D.J. Thorneley R.N.F. Biochemistry. 1996; 35: 9907-9916Crossref PubMed Scopus (35) Google this that both histidine residues are in leading to the C-O bond cleavage. be that the reaction with enzyme is well in the of the experimental and the activity of the mutant is by a of for the H106A and H17A mutant protein, respectively. of the contrast to the monofunctional chorismate synthases from and chorismate synthases have an activity in that NADPH to FMN to its active reduced form Amrhein N. Schmid J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar). the activity of the two histidine mutant in the in the an additional of the amino acid on the to use NADPH This by the of NADPH in the of and of EPSP these both histidine mutant a of NADPH to and for the H17A and H106A mutant protein, respectively, as with enzyme for NADPH were by the from for to and for the H17A and H106A mutant protein, respectively. In reported have provided evidence that NADPH and EPSP for the binding site. a high of EPSP a in the of NADPH P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). This of EPSP is upon of EPSP to chorismate, in a of NADPH the two histidine mutant are EPSP to chorismate, and the of EPSP in contrast to enzyme of FMN to the of oxidized FMN to both histidine mutant by difference and as P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). histidine mutant to suggesting that histidine residue is in binding of the oxidized for and FMN from Ref. from Ref. from Ref. of difference of of of of of of not of from Ref. from Ref. from Ref. P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google of of not in a the upon binding of oxidized FMN to the histidine mutant were to those with enzyme, that the does not the environment of the flavin in the active site in the of EPSP, binding of oxidized FMN to the histidine mutant is that of the two residues plays a role in binding of FMN in the ternary of EPSP to the in the of of EPSP to the H17A and H106A mutant in the of FMN first by The were used to the for In the of the His106 mutant protein, it is to the for protein, whereas it is for the H17A mutant and This that His17 is in EPSP whereas His106 is the the H17A mutant with EPSP are to those reported earlier with the In contrast to the H17A mutant protein, the H106A mutant This finding evidence that His106 is in close proximity to the flavin ring whereas His17 is This to the binding of oxidized FMN which is not by histidine suggesting that binding of EPSP the in the active site such that the flavin ring and the His106 with other in the of substrate. received further support from flavin is as a of EPSP Although the H17A mutant to those with enzyme, the H106A mutant to show this and the by of FMN to the reduced form of FMN is the form in the chorismate synthase catalyzed reaction, have its binding to has been shown that the reduction of FMN by to the reduced form the of a radical species P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of the flavin in the of a of redox the of the redox for the oxidized FMN redox P. Petersen J. Bornemann S. Lowe D.J. Thorneley R.N.F. Biochemistry. 1996; 35: 1643-1652Crossref PubMed Scopus (38) Google Scholar, V. S. Flavins and Scholar). In the of of NcCS, the redox of the to more that of the free in solution Biochem. PubMed Scopus Google This a binding of the reduced form to enzyme as with oxidized FMN, to a of a for reduced FMN of for oxidized FMN P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google histidine mutant more negative redox of and for the H17A and H106A respectively. This a binding of reduced FMN to the two histidine mutant proteins, of 3.3 and of a of chorismate reduced FMN and EPSP are in the a is transiently (6Bornemann S. Lowe D.J. Thorneley R.N.F. Biochemistry. 1996; 35: 9907-9916Crossref PubMed Scopus (35) Google Scholar, P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, P. Amrhein N. P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Lowe D.J. Thorneley R.N.F. J. Chem. Soc. Scopus Google Scholar). This species is within a and all substrate has been Although the leading to this is thought to be the protonation of the position of the reduced form of FMN (pKa of = 6.7 in free its role in catalysis is not the of the histidine residues and both histidine mutant were with to their ability to form this shown in the with both histidine mutant the H17A mutant protein, the is to the with enzyme, an with a in the of less as for enzyme In the for the H106A mutant in to the a in the the of the is with and to the of Thus, these show that both histidine residues the and the of the flavin with the H106A mutant the in of and the of proposed protonation of the position of the reduced flavin in the leading to the of the flavin during the chorismate synthase catalyzed reaction, the this be required for this were in the of on the electron in the isoalloxazine have shown that of in the reduced form have on the in the and the negative and electron J. Chem. Scopus Google Scholar). of the electron in the of reduced a group in position of a this of the protonation state of on the electron in the isoalloxazine ring shown in protonation of a of electron from the position to and of the In of the that a electron transfer from reduced FMN to EPSP initiates C-O bond the of the highest electron in the isoalloxazine ring may be a in The of 5-enolpyruvylshikimate 3-phosphate to chorismate by chorismate synthase is of the flavin-dependent in and mechanistic studies have evidence for a radical mechanism in which the FMN C-O bond by electron to the substrate (6Bornemann S. Lowe D.J. Thorneley R.N.F. Biochemistry. 1996; 35: 9907-9916Crossref PubMed Scopus (35) Google Scholar, 7Macheroux P. Bornemann S. Ghisla S. Thorneley R.N.F. J. Biol. Chem. 1996; 271: 25850-25858Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 9Osborne A. Thorneley R.N.F. Abell C. Bornemann S. J. Biol. Chem. 2000; 275: 35825-35830Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, S. M. Thorneley R.N.F. Abell C. Chem. 2000; PubMed Scopus Google Scholar). The proposed radical for the reaction has been by 2001; PubMed Scopus Google Scholar). the of the first structure of the enzyme in with FMN and EPSP has shown that the substrate in close proximity on the si-face of the flavin isoalloxazine a direct mechanistic role for FMN (10Maclean J. Ali S. Structure. 2003; 11: 1499-1511Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). This structure has provided the first the flavin environment and the amino acid residues in FMN and EPSP of invariant amino acid residues has revealed the of two histidines (12Macheroux P. Schönbrunn E. Svergun D.I. Volkov V.V. Koch M.H.J. Bornemann S. Thorneley R.N.F. Biochem. J. 1998; 335: 319-327Crossref PubMed Scopus (31) Google both of which are found in the active site of chorismate synthase and His106, Scheme The histidine with its is a suitable and common catalyst for by acid-base In with the diethyl that histidines are for catalytic activity by chorismate synthase In with the structure and the of His17 and His106 in all chorismate synthase to this that these two active site histidines are for the by the functional role of the two active site histidines in more have two single mutant with an in position position single mutant proteins, H17A and a 10- and 20-fold net activity for the elimination reaction in with enzyme, respectively, the complete by and the of both histidines in the chorismate synthase reaction. In the of chorismate synthase activity is by of the reduced In the of a bifunctional enzyme, NADPH be utilized in all of the is and reduced FMN is no to The of this on the activity of the both histidine to not only the chorismate synthase this The of NADPH in the H17A H106A with an in the for NADPH and Although the on the of the activity is for the H17A mutant the H106A mutant a more on the to the chorismate synthase activity This that His106 the reduction of oxidized FMN in the active site of the enzyme, of the negative on the reduced flavin of to the This is by the that binding of reduced FMN is in the mutant proteins, in in the H106A mutant The less of the of reduced FMN to the H17A mutant that this residue is in its to a the ability to NADPH for the of the reduced FMN cofactor thought to be to a binding in the bifunctional enzymes not in the monofunctional enzymes Amrhein N. Schmid J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). recent studies have that NADPH a common binding site with EPSP P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In this it is that the of activity of the two histidine to mutant does not to of EPSP to chorismate as a the of EPSP is not as is the with enzyme This to the activity of the bifunctional N. crassa chorismate Although both amino acid the activity of chorismate are apparently not for this enzymatic activity as not the flavin Thus, the amino acid residues that and it be that of the structure of a bifunctional chorismate synthase as to the molecular of The on the catalytic activity the of both histidine residues in Since both mutant are in phosphate His17 and His106 to be in leading to C-O bond of the that both histidine mutant oxidized FMN with a and to those reported for NcCS, that of the two histidine residues is for binding of the oxidized cofactor and studies have shown that the of the ternary of chorismate FMN, and EPSP is with and and were for monofunctional P. Petersen J. Bornemann S. Lowe D.J. Thorneley R.N.F. Biochemistry. 1996; 35: 1643-1652Crossref PubMed Scopus (38) Google Scholar, P. Amrhein N. P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google as well as a bifunctional chorismate synthase P. Amrhein N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, it be that these the of a flavin environment in all chorismate synthases. In contrast to the H17A mutant protein, the and the are in the H106A mutant and that it is the of the flavin ring with this amino acid that is for the the other EPSP binding is only by the H106A whereas the H17A mutant a binding of EPSP and In the structure of the ternary complex, of His17 is within hydrogen bond distance to the phosphate group of EPSP, whereas of His106 is found in a distance to the N(1)-C(2) = O locus of FMN and the group of EPSP (10Maclean J. Ali S. Structure. 2003; 11: 1499-1511Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). it be that of the latter two plays a role in binding the oxidized FMN This is in contrast to which binding for EPSP the difference and the H106A mutant upon of the ternary complex, upon binding of EPSP to the active site. the and the distance His106 and the N(1)-C(2) = O locus of the flavin does not change upon binding of EPSP distance of to is and Å in the and ternary complex, (10Maclean J. Ali S. Structure. 2003; 11: 1499-1511Abstract Full Text Full Text PDF PubMed Scopus (47) Google Thus, it be that the are a direct of to this upon EPSP binding to the si-face of the isoalloxazine The for such an is the which is above the N(1)-C(2) = O locus and within 3 Å of the histidine (10Maclean J. Ali S. Structure. 2003; 11: 1499-1511Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Although the on oxidized FMN is for studies with FMN of which are to be of reduced FMN P. Bornemann S. Ghisla S. Thorneley R.N.F. J. Biol. Chem. 1996; 271: 25850-25858Abstract Full Text Full Text PDF PubMed Scopus (42) Google have binding of the reduced FMN species the species (pKa = 6.7 free in solution (13Dudley K.H. Ehrenberg A. Hemmerich P. Müller F. Helv. Chim. Acta. 1964; 47: 1354-1383Crossref Scopus (153) Google This that upon of a ternary this with reduced the the protonation of by an of its In this His106 (pKa = free in as the acid that the to This role of His106 is by the for the flavin in the of the H106A mutant protein, which the and and Although His106 may the role of a general acid the monoanionic reduced FMN, this does not the pertinent this be required for In earlier studies, have that the of the neutral reduced flavin species in the active site of chorismate synthase may be a of an flavin environment that in serves to the redox of the cofactor as to it a for a putative electron transfer to the substrate P. Bornemann S. Ghisla S. Thorneley R.N.F. J. Biol. Chem. 1996; 271: 25850-25858Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). in of the structure with the imidazole ring of His106 and the group of the substrate the N(1)-C(2) = O locus of the this does not to be the other the protonation on the electron in the of the isoalloxazine ring as shown by Ref. J. Chem. Scopus Google Scholar and in Thus, protonation of may to direct the electron that transfer to the bond of the substrate is In other the the electron in the flavin ring as to catalysis in a earlier cofactor P. Bornemann S. Ghisla S. Thorneley R.N.F. J. Biol. Chem. 1996; 271: 25850-25858Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Based on the structure and with the two histidine mutant a more mechanistic In this binding of EPSP to the reduced FMN an electron is to the substrate C-O bond and the of phosphate with His17 as a general acid to the on the The flavin to a radical species the electron on with of the from by the This step is by the structure of the ternary complex, which that is the only that be for the of the C-(6proR) hydrogen In the of the reduced flavin the state of the cofactor Although this the step as transfer of an it is that a transfer to the reaction. This has been by Ref. 2001; PubMed Scopus Google Scholar and is with the to a flavin radical species during catalytic to this flavin-dependent reaction mechanism are to on the role of other invariant amino acid residues in the active site such as the two residues and that to in the binding of a close to the to of EPSP, suggesting that of the phosphate leaving group is a in the role of the group of EPSP to be of to the reaction mechanism. The of these be by site-directed and the of substrate respectively, and further the mechanism of chorismate are to for the and of

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