Publications

• A new family of 2-hydroxyacid dehydrogenases.

  Grant G.A.

  The NADH-dependent hydroxypyruvate reductase from cucumber and the pdxB gene product of E. coli display significant homology to E. coli D-3-phosphoglycerate dehydrogenase. In contrast, these proteins do not display much similarity with other oxidoreductases or with other 2-hydroxyacid dehydrogenas ... >> More

  The NADH-dependent hydroxypyruvate reductase from cucumber and the pdxB gene product of E. coli display significant homology to E. coli D-3-phosphoglycerate dehydrogenase. In contrast, these proteins do not display much similarity with other oxidoreductases or with other 2-hydroxyacid dehydrogenases in particular. On the basis of their relatedness and the structure of their substrates, these three enzymes constitute a new family of 2-hydroxyacid dehydrogenases distinct from malate and lactate dehydrogenase. << Less

  Biochem Biophys Res Commun 165:1371-1374(1989) [PubMed] [EuropePMC]

• Positive growth rate-dependent regulation of the pdxA, ksgA, and pdxB genes of Escherichia coli K-12.

  Pease A.J., Roa B.R., Luo W., Winkler M.E.

  We found that transcription of the pdxA and pdxB genes, which mediate steps in the biosynthesis of the essential coenzyme pyridoxal 5"-phosphate, and the ksgA gene, which encodes an rRNA modification enzyme and is partly cotranscribed with pdxA, is subject to positive growth rate regulation in Esc ... >> More

  We found that transcription of the pdxA and pdxB genes, which mediate steps in the biosynthesis of the essential coenzyme pyridoxal 5"-phosphate, and the ksgA gene, which encodes an rRNA modification enzyme and is partly cotranscribed with pdxA, is subject to positive growth rate regulation in Escherichia coli K-12. The amounts of the pdxA-ksgA cotranscript and pdxB- and ksgA-specific transcripts and expression from pdxA- and pdxB-lacZ fusions increased as the growth rate increased. The half-lives of ksgA- and pdxB-specific transcripts were not affected by the growth rate, whereas the half-life of the pdxA-ksgA cotranscript was too short to be measured accurately. A method of normalization was applied to determine the amount of mRNA synthesized per gene and the rate of protein accumulation per gene. Normalization removed an apparent anomaly at fast growth rates and demonstrated that positive regulation of pdxB occurs at the level of transcription initiation over the whole range of growth rates tested. RNA polymerase limitation and autoregulation could not account for the positive growth rate regulation of pdxA, pdxB, and ksgA transcription. On the other hand, growth rate regulation of the amount of the pdxA-ksgA cotranscript was abolished by a fis mutation, suggesting a role for the Fis protein. In contrast, the fis mutation had no effect on pdxB- or ksgA-specific transcript amounts. The amounts of the pdxA-ksgA cotranscript and ksgA-specific transcript were repressed in the presence of high intracellular concentrations of guanosine tetraphosphate; however, this effect was independent of relA function for the pdxA-ksgA cotranscript. Amounts of the pdxB-specific transcript remained unchanged during amino acid starvation in wild-type and relA mutant strains. << Less

  J. Bacteriol. 184:1359-1369(2002) [PubMed] [EuropePMC]

• Metabolic relationships between pyridoxine (vitamin B6) and serine biosynthesis in Escherichia coli K-12.

  Lam H.-M., Winkler M.E.

  We propose a pathway leading from erythrose-4-phosphate and glutamate to nitrogen 1 and carbons 5,5', and 6 of the pyridoxine ring. This pathway, which parallels the phosphorylated pathway of serine biosynthesis, is predicted on the homology between PdxB and SerA, the structural similarity between ... >> More

  We propose a pathway leading from erythrose-4-phosphate and glutamate to nitrogen 1 and carbons 5,5', and 6 of the pyridoxine ring. This pathway, which parallels the phosphorylated pathway of serine biosynthesis, is predicted on the homology between PdxB and SerA, the structural similarity between serine and 4-hydroxythreonine, and the possible involvement of SerC in pyridoxine biosynthesis. Several predictions of this hypothetical scheme were tested. Consistent with the proposed pathway, supplement inhibition patterns strongly suggest that SerA enzyme acts in a an alternate pathway of pyridoxine biosynthesis in pdxB mutants. Direct enzyme assays detected erythrose-4-phosphate dehydrogenase activity in crude extracts, which again supports the proposed pathway. Chromosomal insertions in serC caused a requirement for pyridoxine, serine, and aromatic compounds, which directly verified that SerC functions in the pyridoxine biosynthetic pathway. Complementation analysis showed that pdxF and pdxC mutations reported previously are most likely alleles of serC. Growth of serC chromosomal insertion mutants on glycoalaldehyde was found to occur without acquisition of second-site mutations and confirmed that pdxB and serC, but not pdxA, function in the same branch of the pyridoxine pathway. In addition, serC::mini-Mu d insertions revealed that the complex serC-aroA operon lacks internal promoters, that the amino terminus of SerC is not strictly essential for activity, and that antisense transcription occurs in the serC-aroA operon. Growth responses of pdxA, pdxB, and serC mutants to beta-hydroxypyruvate, D-alanine, and glycolate could also be reconciled with the proposed pathway. Finally, the proposed scheme is consistent with previous isotope labeling data and accounts for several other observations about pyridoxine biosynthesis. Together, these physiological and biochemical analyses support the proposed pathway and an evolutionary scenario in which this branch of the pyridoxine pathway evolved from the serine pathway by gene recruitment. << Less

  J. Bacteriol. 172:6518-6528(1990) [PubMed] [EuropePMC]

• A novel alpha-ketoglutarate reductase activity of the serA-encoded 3-phosphoglycerate dehydrogenase of Escherichia coli K-12 and its possible implications for human 2-hydroxyglutaric aciduria.

  Zhao G., Winkler M.E.

  Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. The SerA enzyme is evolutionarily related to the pdxB gene product, 4-phosphoerythronate dehydrogenase, which catalyzes the second step ... >> More

  Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. The SerA enzyme is evolutionarily related to the pdxB gene product, 4-phosphoerythronate dehydrogenase, which catalyzes the second step in one branch of pyridoxal 5'-phosphate coenzyme biosynthesis. Both the Ser and pyridoxal 5'-phosphate biosynthetic pathways use the serC(pdxF)-encoded transaminase in their next steps. In an analysis of these parallel pathways, we attempted to couple the transaminase and dehydrogenase reactions in the reverse direction. Unexpectedly, we found that the SerA enzyme catalyzes a previously undetected reduction of alpha-ketoglutarate (alpha KG) to 2-hydroxyglutaric acid (HGA). Numerous criteria ruled out the possibility that this SerA alpha KG reductase activity was caused by contamination in the substrate or purified enzyme preparations. HGA was confirmed as the product of the SerA alpha KG reductase reaction by thin-layer chromatography and by enzyme assays showing that both the D- and L-isomers of HGA were substrates for the reverse (dehydrogenase) reaction. Detailed steady-state kinetic analyses showed that alpha KG reduction (apparent Michaelis-Menten constant [Km(app)] = 88 microM; apparent catalytic constant [kcat(app)] = 33.3 s-1) and 3-phosphohydroxypyruvate reduction (Km(app) = 3.2 microM; kcatapp = 27.8 s-1), which is the reverse reaction of 3PG oxidation, were the major in vitro activities of the SerA enzyme. The SerA alpha KG reductase was inhibited by Ser, D-HGA, 3PG, and glycine (Gly), whereas the D-HGA dehydrogenase was inhibited by Ser, alpha KG, 3-phosphohydroxypyruvate, and Gly. The implications of these findings for the regulation of Ser biosynthesis, the recycling of NADH, and the enzymology of 2-hydroxyacid dehydrogenases are discussed. Since the same pathway of Ser biosynthesis seems to be present in all organisms, these results suggest that a mutation in the human SerA homolog may contribute to the neurometabolic diseases D- and L-2-hydroxyglutaric aciduria, which lead to the accumulation of D-HGA and L-HGA, respectively. << Less

  J. Bacteriol. 178:232-239(1996) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Divergent transcription of pdxB and homology between the pdxB and serA gene products in Escherichia coli K-12.

  Schoenlein P.V., Roa B.B., Winkler M.E.

  We report the DNA sequence and in vivo transcription start of pdxB, which encodes a protein required for de novo biosynthesis of pyridoxine (vitamin B6). The DNA sequence confirms results from previous minicell experiments showing that pdxB encodes a 41-kilodalton polypeptide. RNase T2 mapping of ... >> More

  We report the DNA sequence and in vivo transcription start of pdxB, which encodes a protein required for de novo biosynthesis of pyridoxine (vitamin B6). The DNA sequence confirms results from previous minicell experiments showing that pdxB encodes a 41-kilodalton polypeptide. RNase T2 mapping of in vivo transcripts and corroborating experiments with promoter expression vector pKK232-8 demonstrated that the pdxB promoter shares its -10 region with an overlapping, divergent promoter. Thus, pdxB must be the first gene in the complex pdxB-hisT operon. The steady-state transcription level from these divergent promoters, which probably occlude each other, is approximately equal in bacteria growing in rich medium at 37 degrees C. The divergent transcript could encode a polypeptide whose amino-terminal domain is rich in proline and glutamine residues. Similarity searches of protein data bases revealed a significant number of amino acid matches between the pdxB gene product and D-3-phosphoglycerate dehydrogenase, which is encoded by serA and catalyzes the first step in the phosphorylated pathway of serine biosynthesis. FASTA and alignment score analyses indicated that PdxB and SerA are indeed homologs and share a common ancestor. The amino acid alignment between PdxB and SerA implies that PdxB is a 2-hydroxyacid dehydrogenase and suggests possible NAD+, substrate binding, and active sites of both enzymes. Furthermore, the fact that 4-hydroxythreonine, a probable intermediate in pyridoxine biosynthesis, is structurally related to serine strongly suggests that the pdxB gene product is erythronate-4-phosphate dehydrogenase. The homology between PdxB and SerA provides considerable support for Jensen's model of enzyme recruitment as the basis for the evolution of different biosynthetic pathways. << Less

  J. Bacteriol. 171:6084-6092(1989) [PubMed] [EuropePMC]