RESUMO
The beta-hydroxyacid dehydrogenases are a structurally conserved family of enzymes that catalyze the NAD(+) or NADP(+)-dependent oxidation of specific beta-hydroxyacid substrates like beta-hydroxyisobutyrate. These enzymes share distinct domains of amino acid sequence homology, most of which now have assigned putative functions. 6-phosphogluconate dehydrogenase and beta-hydroxyisobutyrate dehydrogenase, the most well-characterized members, both appear to be readily inactivated by chemical modifiers of lysine residues, such as 2,4,6-trinitrobenzene sulfonate (TNBS). Peptide mapping by ESI-LCMS showed that inactivation of beta-hydroxyisobutyrate dehydrogenase with TNBS occurs with the labeling of a single lysine residue, K248. This lysine residue is completely conserved in all family members and may have structural importance relating to cofactor binding. The structural framework of the beta-hydroxyacid dehydrogenase family is shared by many bacterial homologues. One such homologue from E. coli has been cloned and expressed as recombinant protein. This protein was found to have enzymatic activity characteristic of tartronate semialdehyde reductase, an enzyme required for bacterial biosynthesis of D-glycerate. A homologue from H. influenzae was also cloned and expressed as recombinant protein. This protein was active in the oxidation of D-glycerate, but showed approximately ten-fold higher activity with four carbon substrates like beta-D-hydroxybutyrate and D-threonine. This enzyme might function in H. influenzae, and other species, in the utilization of polyhydroxybutyrates, an energy storage form specific to bacteria. Cloning and characterization of these bacterial beta-hydroxyacid dehydrogenases extends our knowledge of this enzyme family.
Assuntos
Desidrogenases de Carboidrato/química , Desidrogenases de Carboidrato/metabolismo , Sequência de Aminoácidos , Desidrogenases de Carboidrato/genética , Sequência Conservada , Escherichia coli/enzimologia , Escherichia coli/genética , Haemophilus influenzae/enzimologia , Haemophilus influenzae/genética , Lisina/química , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
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.
