Crystal Structure of Escherichia coli PdxA, an Enzyme Involved in the Pyridoxal Phosphate Biosynthesis Pathway

Pyridoxal 5′-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-l-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP+ or NAD+ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn2+. The protein forms tightly bound dimers. Each monomer has an α/β/α-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn2+ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp247 and Asp267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP. Pyridoxal 5′-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-l-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP+ or NAD+ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn2+. The protein forms tightly bound dimers. Each monomer has an α/β/α-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn2+ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp247 and Asp267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP. Pyridoxal 5′-phosphate, the catalytically active form of vitamin B6, is an important cofactor for many enzymes responsible for the metabolic conversions of amino acids. Vitamin B6 (pyridoxine) and its derivatives are also efficient singlet oxygen quenchers and potent fungal antioxidants (1Bilski P. Li M.Y. Ehrenshaft M. Daub M.E. Chignell C.F. Photochem. Photobiol. 2000; 71: 129-134Crossref PubMed Scopus (291) Google Scholar). Two different pathways for de novo synthesis of pyridoxine are now recognized. One of these, found in all Archaea, eukaryotes, and in some bacteria, uses the singlet oxygen resistance (SOR1(Pdx1)) gene product, a highly conserved enzyme (2Ehrenshaft M. Bilski P. Li M.Y. Chignell C.F. Daub M.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9374-9378Crossref PubMed Scopus (261) Google Scholar). A number of eubacteria, including Escherichia coli, utilize a specific pathway for pyridoxal phosphate synthesis that is distinct and has been characterized for some time (3Hill R.E. Spenser I.D. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. ASM Press, Washington D. C.1996: 695-703Google Scholar). Analysis of sequences from several genomes has revealed that organisms may encode either the SOR1 or E. coli-like pyridoxine biosynthesis genes, but not both (4Ehrenshaft M. Chung K.R. Jenns A.E. Daub M.E. Curr. Genet. 1999; 34: 478-485Crossref PubMed Scopus (41) Google Scholar). The pyridoxal 5′-phosphate biosynthesis pathway has been well characterized in E. coli, and consists of six enzymatic steps, beginning with erythrose 4-phosphate and deoxyxylulose 5-phosphate (5Yang Y. Zhao G. Man T.K. Winkler M.E. J. Bacteriol. 1998; 180: 4294-4299Crossref PubMed Google Scholar, 6Laber B. Maurer W. Scharf S. Stepusin K. Schmidt F.S. FEBS Lett. 1999; 449: 45-48Crossref PubMed Scopus (90) Google Scholar, 7Mittenhuber G. J. Mol. Microbiol. Biotechnol. 2001; 3: 1-20PubMed Google Scholar). All of the enzymes in this pathway have been identified. Of the six enzymes, PdxA, 1The abbreviations used are: PdxA, 4-hydroxythreonine-4-phosphate dehydrogenase; HTP, 4-hydroxy-l-threonine phosphate; PdxJ, pyridoxine-5′-phosphate synthase; r.m.s., root mean suare. erythromate-4-phosphate dehydrogenase (PdxB), and pyridoxine-5′-phosphate oxidase are unique to this pathway (5Yang Y. Zhao G. Man T.K. Winkler M.E. J. Bacteriol. 1998; 180: 4294-4299Crossref PubMed Google Scholar), whereas SerC and GapA also function in other biosynthetic pathways (5Yang Y. Zhao G. Man T.K. Winkler M.E. J. Bacteriol. 1998; 180: 4294-4299Crossref PubMed Google Scholar). The fourth step in this pathway is catalyzed by PdxA (E.C. 1.1.1.262), which converts 4-hydroxy-l-threonine 4-phosphate (HTP) to 3-amino-1-hydroxyacetone 1-phosphate, an oxidative decarboxylation that may involve 2-amino-3-keto-4-hydroxybutyric acid 4-phosphate as an intermediate (8Cane D.E. Hsiung Y. Cornish J.A. Robinson J.K. Spenser I.D. J. Am. Chem. Soc. 1998; 120: 1936-1937Crossref Scopus (58) Google Scholar) (Scheme 1). Either NADP+ or NAD+ function as cofactors, whereas the free alcohol 4-hydroxy-l-threonine is not a substrate for the reaction (8Cane D.E. Hsiung Y. Cornish J.A. Robinson J.K. Spenser I.D. J. Am. Chem. Soc. 1998; 120: 1936-1937Crossref Scopus (58) Google Scholar). The product of the PdxA reaction is used along with deoxyxylulose 5-phosphate by pyridoxine-5′-phosphate synthase (PdxJ) to generate pyridoxine 5′-phosphate along with inorganic phosphate. This key enzyme functions in closure of the aromatic pyridoxine ring (6Laber B. Maurer W. Scharf S. Stepusin K. Schmidt F.S. FEBS Lett. 1999; 449: 45-48Crossref PubMed Scopus (90) Google Scholar, 9Cane D.E. Du S. Robinson J.K. Hsiung Y. Spenser I.D. J. Am. Chem. Soc. 1999; 121: 7722-7723Crossref Scopus (78) Google Scholar, 10Cane D.E. Du S. Spenser I.D. J. Am. Chem. Soc. 2001; 122: 4213-4214Crossref Scopus (29) Google Scholar). Treatment of purified PdxA with 1 mm EDTA abolishes oxidation of HTP, suggesting the presence of a tightly bound divalent metal ion. Addition of 1 mm Mn2+, Co2+, Mg2+, or Ca2+ restores full activity (9Cane D.E. Du S. Robinson J.K. Hsiung Y. Spenser I.D. J. Am. Chem. Soc. 1999; 121: 7722-7723Crossref Scopus (78) Google Scholar), whereas 1 mm Ni2+ or 2 mm Zn2+ restored half of the original PdxA activity. Although the product of the reaction, 3-amino-1-hydroxyacetone 1-phosphate, undergoes facile dimerization in the absence of PdxJ and the co-substrate deoxyxylulose phosphate, we have directly detected 3-amino-1-hydroxyacetone 1-phosphate by electrospray ionization mass spectrometry of PdxA incubation mixtures. 2J. Banks and D. E. Cane, unpublished observations. None of the putative intermediate, 2-amino-3-oxo-4-hydroxybutyric acid 4-phosphate, could be detected in the reaction mixture, even as early as 30 s after initiation of the reaction, suggesting that if this compound is formed, it never leaves the PdxA active site. In principle, PdxA might catalyze either a stepwise or a concerted oxidative decarboxylation of HTP. For example, threonine dehydrogenase catalyzes the biochemically similar, reversible NAD-dependent oxidation of l-threonine to l-2-amino-3-ketobutyrate (Scheme 2) (11Marcus J.P. Dekker E.E. J. Bacteriol. 1993; 175: 6505-6511Crossref PubMed Scopus (32) Google Scholar). The product, l-2-amino-3-ketobutyrate, which is normally converted to glycine and acetyl-CoA by 2-amino-3-ketobutyrate CoA ligase, can undergo pH-dependent decarboxylation, with a half-life ranging from 8.6 min at pH 5.9 to 140 min at pH 11.1 (12Marcus J.P. Dekker E.E. Biochem. Biophys. Res. Commun. 1993; 190: 1066-1072Crossref PubMed Scopus (17) Google Scholar). PdxA and threonine dehydrogenase show no significant amino acid sequence similarity. By contrast, use of the PSI-BLAST sequence comparison algorithm (13Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (61090) Google Scholar) reveals ∼30–35% sequence identity over a short ∼25 residue segment between E. coli PdxA and both isocitrate dehydrogenase and 3-isopropylmalate dehydrogenases (9Cane D.E. Du S. Robinson J.K. Hsiung Y. Spenser I.D. J. Am. Chem. Soc. 1999; 121: 7722-7723Crossref Scopus (78) Google Scholar). Interestingly, each of the latter two enzymes catalyzes a nicotinamide- and divalent metal ion-dependent oxidative decarboxylation of a β-hydroxy acid substrate (Scheme 2). Significantly, the isocitrate dehydrogenase reaction has been shown to proceed by a stepwise mechanism that involves the corresponding 3-keto acid, oxalosuccinate intermediate, based on multiple isotope effect (2H, 13C) studies, as well as the ability of isocitrate dehydrogenase to catalyze both the decarboxylation and the reduction of oxalosuccinate, with a ∼10-fold preference for decarboxylation (14Grissom C.B. Cleland W.W. Biochemistry. 1988; 27: 2934-2943Crossref PubMed Scopus (42) Google Scholar). Structural characterization of the enzymes of the pyridoxine biosynthetic pathway has so far been limited to E. coli PdxJ (15Franco M.G. Laber B. Huber R. Clausen T. Structure (Camb.). 2001; 9: 245-253Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 16Garrido-Franco M. Ehlert S. Messerschmidt A. Marinkovic S. Huber R. Laber B. Bourenkov G.P. Clausen T. J. Biol. Chem. 2002; 277: 12396-12405Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 17Yeh J.I. Du S. Pohl E. Cane D.E. Biochemistry. 2002; 41: 11649-11657Crossref PubMed Scopus (14) Google Scholar) and pyridoxine-5′-phosphate oxidase from Saccharomyces cerevisiae (Protein Data Bank number 1CIO), the enzyme that oxidizes pyridoxine 5′-phosphate and pyridoxamine 5′-phosphate to pyridoxal 5′-phosphate. The x-ray structure of PdxA bound to Zn2+ as well as the HTP presented here reveals a protein that can be divided into two subdomains, and which shares structural similarities with both isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and, in addition, sequence conservation between PdxA and the two dehydrogenases of residues involved in the cofactor binding argue for a similar manner of nicotinamide cofactor binding and similar, stepwise, biochemical mechanisms for oxidative decarboxylation. Furthermore, based on the complex with HTP in the presence of Zn2+, sequence conservation analysis among PdxA enzymes from different sources, and structural comparison with the isocitrate dehydrogenase family, the location of the PdxA active site is stipulated to be in a cleft between the two subdomains and at the interface between the two molecules of the dimer, a of conserved amino acids. and gene into a of the a site to an with a into the E. coli for protein J. 9: PubMed Scopus Google Scholar). The at to an A of in with of A with and the at for an by and in of mm pH mm mm of by on for a of 30 s with s between each for The by 30 The protein on a in and the This protein to a acid with The with mm pH mm and bound with the mm PdxA as a on both and a (Protein at a protein of and at and Data crystal forms of PdxA with the protein by A of 2 of protein in mm pH mm with of pH mm mm pH and over the to a of in 1 These are group with a and two molecules in the These to from the protein mm pH mm and mm as a These to the group with a and molecules in the These to in a of with in a and at in a Data at a All including the Z. W. 1997; PubMed Scopus Google and of for crystal with HTP the form of form crystal form of the on the crystal free free but of and of the unique not in the for HTP complex and crystal form from PdxA crystal with HTP the form of crystal form of the on the crystal free but of and of the unique not in the for HTP complex and crystal form in a Structure and of form used for structure For the from to of the within the found the J. Biol. 1999; PubMed Scopus Google Scholar). The an of of of the by the Biol. 2001; PubMed Scopus (78) Google Scholar), in an of the of to The of with of the of the the M. A. PubMed Scopus Google Scholar). of with the P. J. M. T. Biol. 1998; PubMed Scopus Google Scholar) in the with an of free used in the early of but as the used are in The residues for each Zn2+, and with the well residue in each monomer The residues from the site as well as 1The abbreviations used are: PdxA, 4-hydroxythreonine-4-phosphate dehydrogenase; HTP, 4-hydroxy-l-threonine phosphate; PdxJ, pyridoxine-5′-phosphate synthase; r.m.s., root mean suare. not have and not The structure of PdxA from form by the the J. A. Scopus Google Scholar). The the structure of the dimer of PdxA from the form molecules in the from the two of the function The of and of the are in The root mean between the two crystal form of PdxA is for all the of the Structure of the of the complex of PdxA with a form crystal in mm HTP for A to the corresponding to the HTP product in the and the as for the PdxA for PdxA form and and the complex of form with HTP have been with the for Structural with Data Bank and Structure of the PdxA structure of PdxA from E. coli by the and to a of free at for the crystal form of PdxA, of free for the crystal form of PdxA at and an of free for the complex of PdxA with HTP at These have been with for the from an analysis J. 1993; Google Scholar) for the over of residues in the E. coli PdxA has an with a on both by The within this is with the six by two and in the to the This is along its to the by so that the and in the 2). are to the of the whereas three along the other of the in its Although is no of the could be divided into two subdomains, with residues and 1 and The and are within 1 and are in to each by The site is located the of the at the interface between the two subdomains, on the with The of PdxA is to that found in the dehydrogenases J.L. D.E. PubMed Scopus Google Scholar, K. M. Y. Y. T. J. Mol. Biol. PubMed Scopus Google Scholar) as in the Structural of B. Nucleic Acids Res. 2000; PubMed Scopus Google of the of the PdxA monomer the of the and 1 is in and the 2 in The and are Structure of the PdxA that the PdxA molecules form in This is in to an PdxA characterized as based on (8Cane D.E. Hsiung Y. Cornish J.A. Robinson J.K. Spenser I.D. J. Am. Chem. Soc. 1998; 120: 1936-1937Crossref Scopus (58) Google Scholar). The presence of is with the in the crystal with each dimer of The two of the dimer are by a to the a The of the two in the are very similar, root mean of for all of subdomains 1 or 2 an of that is a in the of the two subdomains to in the The dimer is the interactions of subdomains 2 of the PdxA monomers. These interactions are by the from each monomer and from residues in the and are a of including and a of Analysis of the multiple sequence for enzymes sequence similarity to PdxA number reveals that the residues found at the dimer interface are well suggesting that the mode of dimerization is to all of this A of Zn2+ is at the dimer interface, with residues from both to the coordination for each Zn2+ site The dimer a is or of the of each and Structural from E. coli and its form a conserved of enzymes, and in the of G. J. Mol. Microbiol. Biotechnol. 2001; 3: 1-20PubMed Google Scholar). analysis PSI-BLAST (13Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (61090) Google Scholar) sequences in and similar sequence in the The sequence identity between E. coli PdxA and the from for Salmonella to for is sequence identity between the E. coli PdxA and that from The conserved residues in the and as well as at the dimerization interface Structural comparison of E. coli PdxA with other protein the Nucleic Acids Res. 1997; 25: PubMed Scopus Google Scholar). the structurally similar found to be isocitrate dehydrogenase (Protein Data Bank of for and isopropylmalate dehydrogenase (Protein Data Bank for The limited amino acid sequence similarity between PdxA and the other two from PSI-BLAST as has been (9Cane D.E. Du S. Robinson J.K. Hsiung Y. Spenser I.D. J. Am. Chem. Soc. 1999; 121: 7722-7723Crossref Scopus (78) Google Scholar). all three catalyze of acids. The of PdxA with the two dehydrogenases that and are structurally and in of PdxA, from the can be on with an of 1997; PubMed Scopus Google Furthermore, the dimerization interface of PdxA and the other two dehydrogenases involves the of the and is similar Structural residues of for PdxA to all three sequences Zn2+ the of the protein are two corresponding to metal on the coordination and the of we have as two Zn2+ no Zn2+ of the metal have been in E. coli and with the enzyme the and The presence of a tightly bound divalent metal has been by Cane (9Cane D.E. Du S. Robinson J.K. Hsiung Y. Spenser I.D. J. Am. Chem. Soc. 1999; 121: 7722-7723Crossref Scopus (78) Google Scholar) as The Zn2+ are at the dimer interface and are coordinated by and from monomer and from the The coordination of each Zn2+ is by three molecules The between the Zn2+ and the or from to These three are conserved in all amino acid sequences that all enzymes from this are enzymes The coordination of each Zn2+ ion by conserved histidine residues from both of the dimer that dimerization is essential for enzymatic activity. have the in a mm HTP. The a substrate bound to of the two molecules of the PdxA dimer The HTP in a in the of the One of this is by residues from the monomer of the dimer, which converts it into a and cleft of the two molecules of the dimer that in the of the two subdomains. The of HTP is bound to the monomer that has a In the of PdxA the monomer an inorganic phosphate ion in the as the phosphate group of HTP. The of this cleft in the of that to the phosphate phosphate of HTP substrate bound to the in the corresponding to the bound HTP in the active site of the PdxA at 2 The residues in the of the HTP and Zn2+ from the of of the dimer the substrate binding site, with the HTP shown in Each is The of NADP+ from the structure of isocitrate PdxA and as shown in the HTP and the coordination of Zn2+ ion. a of the active site of PdxA with bound The cofactor as in show the between and and the between of and of The HTP the phosphate as well as the three molecules that the Zn2+ coordination The phosphate group of the HTP the inorganic phosphate whereas the and the in a Zn2+ ion In this complex the and are to a to the of the and This also the for a between the of the HTP and of and between of HTP and the of both and the latter from the other of the In addition, the group of HTP is to the conserved residue and the phosphate group is to in of as well as to the of found within the highly conserved sequence PdxA enzyme NAD+ or NADP+ as the cofactor for oxidation of HTP. Although we of PdxA in the presence of to mm of either of cofactors, with and HTP, in no could corresponding to the nicotinamide cofactor be found in the to the into the also This may that HTP normally this has to be Although we not directly the of the cofactor in PdxA, its can be by a comparison with the structure of the E. coli isocitrate dehydrogenase with and (Protein Data Bank D.E. Biochemistry. 2001; PubMed Scopus Google Scholar). In this structure the of the NADP+ cofactor the segment of the a and the and nicotinamide into on either of the The with on and on the whereas the nicotinamide between and the The of the is on the of the The corresponding of the structure of PdxA and the very well on that of isocitrate dehydrogenase an segment of is also Furthermore, residues with the residue are conserved in the sequences from the PdxA We the of PdxA with HTP, and the isocitrate dehydrogenase with (Protein Data Bank the structure of the complex with a of NADP+ from the isocitrate dehydrogenase to the structure that binding of the HTP substrate a of between the subdomains and that the of the PdxA is essential for substrate binding and product the structure of the complex that the Zn2+ coordinated by three conserved an essential role in substrate binding and that the phosphate group of HTP to this The role of the phosphate group in HTP binding a structural for the that the free alcohol 4-hydroxy-l-threonine is not a substrate for PdxA (8Cane D.E. Hsiung Y. Cornish J.A. Robinson J.K. Spenser I.D. J. Am. Chem. Soc. 1998; 120: 1936-1937Crossref Scopus (58) Google Scholar). and in the of the is on the of the NADP+ and with the the of PdxA for either NAD+ or The nicotinamide ring of the if as in isocitrate be in a to the bound substrate HTP with the of its from the of HTP that is to be oxidation The of in the is with this By a of has been for the complex of isocitrate dehydrogenase D.E. 1997; 277: PubMed Scopus Google Scholar). with Zn2+ the a of the hydroxyl similar to that in other metal ion-dependent Furthermore, of HTP is within to the of and which are at the pH of the from this hydroxyl could be by of the and to the as are on the of the in addition a to the conserved Asp267, which its for coordination of the Zn2+ ion. of Asp247 may be by its with and, a with and of the to the of the and at the time to with and isocitrate and isopropylmalate dehydrogenase all catalyze nicotinamide- and divalent metal ion-dependent oxidative of acids. isocitrate dehydrogenase and and isopropylmalate dehydrogenase have all been shown to utilize the of the nicotinamide corresponding to the for PdxA (14Grissom C.B. Cleland W.W. Biochemistry. 1988; 27: 2934-2943Crossref PubMed Scopus (42) Google Scholar). In to PdxA, both isocitrate dehydrogenase and isopropylmalate dehydrogenase utilize of Zn2+. The comparison of the that the location of the is not the in enzymes. the three that Zn2+ in PdxA, and are conserved in all PdxA enzymes, are not in the other two In of PdxA have shown that of for Zn2+ in a in which metal is in the protein (9Cane D.E. Du S. Robinson J.K. Hsiung Y. Spenser I.D. J. Am. Chem. Soc. 1999; 121: 7722-7723Crossref Scopus (78) Google Scholar). the divalent metal in both isocitrate dehydrogenase and isopropylmalate dehydrogenase is between the oxygen of the hydroxyl and of the and isopropylmalate are in with a between the and K. K. K. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), in to the for the corresponding in HTP. the and are with a mechanism in which the in the 3-keto intermediate is to the of the as for the decarboxylation is significant that isocitrate dehydrogenase has been shown to catalyze a stepwise oxidative decarboxylation the of oxalosuccinate (14Grissom C.B. Cleland W.W. Biochemistry. 1988; 27: 2934-2943Crossref PubMed Scopus (42) Google Scholar). the substrate binding of isocitrate dehydrogenase and isopropylmalate dehydrogenase are highly with the bound by interactions with active site and residues and to the hydroxyl group of a K. K. K. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), similar to the interactions of the of HTP with and as well as a bound of We for the gene into an E. coli and to J. D. for on the

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