Novel beta-hydroxyacid dehydrogenases in Escherichia coli and Haemophilus influenzae

Our laboratory has previously reported a structurally and mechanistically r elated family of beta -hydroxyacid dehydrogenases with significant homology to beta -hydroxyisobutyrate dehydrogenase. A large number of the members o f this family are hypothetical proteins of bacterial origin with unknown id entity in terms of their substrate specificities and metabolic roles. The E scherichia coli beta -hydroxyacid dehydrogenase homologue corresponding to the AE000157 locus was cloned and expressed with a 6-histidine tag for spec ific purification. The purified recombinant protein very specifically catal yzed the NAD(+)-dependent oxidation of D-glycerate and the NADH-dependent r eduction of tartronate semialdehyde, identifying this protein as a tartrona te semialdehyde reductase. Further evidence for identification as tartronat e 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 semiald ehyde, producing an operon clearly designed for D-glycerate biosynthesis fr om tartronate semialdehyde. The single beta -hydroxyacid dehydrogenase homo logue from Haemophilus influenzae was also cloned, expressed, and purified with a g-histidine tag. This protein also catalyzed the NAD(+)-dependent ox idation of D-glycerate but was significantly more efficient in the oxidatio n of four-carbon beta -hydroxyacids like D-hydroxybutyrate and D-threonine. This enzyme differs from all the presently known beta -hydroxybutyrate deh ydrogenases which are well established members of the short chain dehydroge nase/reductase superfamily.