NAD-DEPENDENT D-2-HYDROXYISOCAPROATE DEHYDROGENASE OF LACTOBACILLUS-DELBRUECKII SUBSP BULGARICUS - GENE CLONING AND ENZYME CHARACTERIZATION()

A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for bo th lactate dehydrogenase and pyruvate formate lyase, and thus unable t o grow anaerobically. One recombinant clone was found to display a bro ad specificity NAD(+)-dependent D-2-hydroxyacid dehydrogenase activity . The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333-res idue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy -4-methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus case i and other NAD(+)-dependent D-lactate dehydrogenases (D-LDH) from sev eral other bacterial species. The hdhD gene was overexpressed under th e control of the lambda phage P-L promoter and the enzyme was purified with a two-step method. The L. delbrueckii subsp. bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2-o xoacid substrates except those having a branched beta-carbon.