Biochemical characterization of gapB-encoded erythrose 4-phosphate dehydrogenase of Escherichia coli K-12 and its possible role in pyridoxal 5'-phosphate biosynthesis

Abstract

One step in de novo pyridoxine (vitamin B 6 ) and pyridoxal 5-phosphate biosynthesis was predicted to be an oxidation catalyzed by an unidentified D-erythrose-4-phosphate dehydrogenase (E4PDH). To help identify this E4PDH, we purified the Escherichia coli K-12 gapA-and gapB-encoded dehydrogenases to homogeneity and tested whether either uses D-erythrose-4-phosphate (E4P) as a substrate. gapA (gap1) encodes the major D-glyceraldehyde-3-phosphate dehydrogenase (GA3PDH). The function of gapB (gap2) is unknown, although it was suggested that gapB encodes a second form of GA3PDH or is a cryptic gene. We found that the gapBencoded enzyme is indeed an E4PDH and not a second GA3PDH, whereas gapA-encoded GA3PDH used E4P poorly, if at all, as a substrate under the in vitro reaction conditions used in this study. The amino terminus of purified E4PDH matched the sequence predicted from the gapB DNA sequence. Purified E4PDH was a heat-stable tetramer with a native molecular mass of 132 kDa. ؊1 in steady-state reactions in which NADH formation was determined. From specific activities in crude extracts, we estimated that there are at least 940 E4PDH tetramer molecules per bacterium growing in minimal salts medium plus glucose at 37؇C. Thin-layer chromatography confirmed that the product of the E4PDH reaction was likely the aldonic acid 4-phosphoerythronate. To establish a possible role of E4PDH in pyridoxal 5-phosphate biosynthesis, we showed that 4-phosphoerythronate is a likely substrate for the 2-hydroxy-acid dehydrogenase encoded by the pdxB gene. Implications of these findings in the evolution of GA3PDHs are also discussed. On the basis of these results, we propose renaming gapB as epd (for D-erythrose-4-phosphate dehydrogenase).

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

“…We overexpressed SerA 3PG dehydrogenase from strain JM105 containing the pSAWT (P tac -serA ϩ ) expression vector (34) and purified the SerA enzyme by the scheme outlined in Table 1 and Materials and Methods. We also purified His 6 -SerC(PdxF) 3PHP transaminase as described before (46). We attempted to couple the SerC(PdxF) and SerA reactions in the reverse direction by first mixing ␣KG, NADH, and the purified SerA and His 6 -SerC(PdxF) enzymes (Fig.…”

“…(46,47) because overexpressed SerA amounted to about 30% of the total protein in E. coli crude extracts as determined by SDS-PAGE (data not shown). Beyond the DEAE-cellulose I step, the purification mainly removed minor contaminating proteins.…”

“…As in the case of the SerA 3PG dehydrogenase, the forward reaction catalyzed by PdxB 4PE dehydrogenase does not readily occur in vitro. To date, we have been able to detect only a single turnover of 4PE by the purified PdxB enzyme in the forward dehydrogenase direction (25,46). The third step in this branch of pyridoxine 5Ј-phosphate and pyridoxal 5Ј-phosphate biosynthesis is catalyzed by the same SerC(PdxF) transaminase that catalyzes the second step in the Ser pathway (Fig.…”

“…SerA 3PG dehydrogenase is a member of a family of evolutionarily related 2-hydroxyacid dehydrogenases that includes the pdxB-encoded 4-phosphoerythronate (4PE) dehydrogenase (15,33,40,41). 4PE dehydrogenase catalyzes the second committed step in one branch of pyridoxine 5Ј-phosphate and pyridoxal 5Ј-phosphate coenzyme biosynthesis in E. coli (20,25,33,46). As in the case of the SerA 3PG dehydrogenase, the forward reaction catalyzed by PdxB 4PE dehydrogenase does not readily occur in vitro.…”

Isolation of a pdxJ point mutation that bypasses the requirement for the PdxH oxidase in pyridoxal 5' -phosphate coenzyme biosynthesis in Escherichia coli K-12

“…We prepared a genomic library of DNA from the suppressor mutant in vector pBR322 (4,38). Transformants of the library that suppressed the ⌬pdxH::⍀(Cm r ) mutation in TX2768 were selected on MM-glucose plates containing antibiotics (25.0 g of ampicillin and 12.5 g of chloramphenicol per ml) at 37ЊC in the absence of oxygen.…”