Abstract
Our laboratory has previously reported a structurally and mechanistically related family of beta-hydroxyacid dehydrogenases with significant homology to beta-hydroxyisobutyrate dehydrogenase. A large number of the members of this family are hypothetical proteins of bacterial origin with unknown identity in terms of their substrate specificities and metabolic roles. The Escherichia coli beta-hydroxyacid dehydrogenase homologue corresponding to the locus was cloned and expressed with a 6-histidine tag for specific purification. The purified recombinant protein very specifically catalyzed the NAD(+)-dependent oxidation of d-glycerate and the NADH-dependent reduction of tartronate semialdehyde, identifying this protein as a tartronate semialdehyde reductase. Further evidence for identification as tartronate semialdehyde reductase is the observation that the coding region for this protein is directly preceded by genes coding for hydroxypyruvate isomerase and glyoxylate carboligase, two enzymes that synthesize tartronate semialdehyde, producing an operon clearly designed for d-glycerate biosynthesis from tartronate semialdehyde. The single beta-hydroxyacid dehydrogenase homologue from Haemophilus influenzae was also cloned, expressed, and purified with a 6-histidine tag. This protein also catalyzed the NAD(+)-dependent oxidation of d-glycerate but was significantly more efficient in the oxidation of four-carbon beta-hydroxyacids like d-hydroxybutyrate and d-threonine. This enzyme differs from all the presently known beta-hydroxybutyrate dehydrogenases which are well established members of the short chain dehydrogenase/reductase superfamily.
Highlights
The -hydroxyacid dehydrogenases are a structurally and mechanistically related family of enzymes that includes -hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, a novel D-phenylserine dehydrogenase, and numerous unidentified bacterial homologues [1, 2]
The rat enzyme is highly specific for S--hydroxyisobutyrate but is active in the NADϩ-dependent oxidation of L-glycerate, L-serine, and R--hydroxyisobutyrate, with relatively high Km values compared with S--hydroxyisobutyrate. 6-Phosphogluconate dehydrogenase is highly specific for NADPϩ-dependent oxidation of 6-phospho-gluconate and has well known metabolic roles as a part of the pentose phosphate pathway
We report five distinct -hydroxyacid dehydrogenase homologues in the Escherichia coli genome, including a single gene for 6-phosphogluconate dehydrogenase
Summary
The -hydroxyacid dehydrogenases are a structurally and mechanistically related family of enzymes that includes -hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, a novel D-phenylserine dehydrogenase, and numerous unidentified bacterial homologues [1, 2]. A highly conserved domain with a consensus sequence of DAPVSGG appears to be structurally and functionally important to -hydroxyacid substrate binding in 6-phosphogluconate dehydrogenase [3] and most likely in other family members. We first hypothesized that one or more of these homologues may correspond to tartronate semialdehyde reductase, a -hydroxyacid dehydrogenase type of reaction that functions in bacterial biosynthesis of glycerate from glyoxylate through the action of glyoxylate carboligase [1].
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