Bernard N, Johnsen K, Ferain T, Garmyn D, Hols P, Holbrook J J, Delcour J
Unité de Génétique, Université Catholique de Louvain, Belgium.
Eur J Biochem. 1994 Sep 1;224(2):439-46. doi: 10.1111/j.1432-1033.1994.00439.x.
A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and thus unable to grow anaerobically. One recombinant clone was found to display a broad 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-residue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy-4-methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus casei and other NAD(+)-dependent D-lactate dehydrogenases (D-LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage PL 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-oxoacid substrates except those having a branched beta-carbon.
使用德氏乳杆菌保加利亚亚种的基因组文库来互补一株缺乏乳酸脱氢酶和丙酮酸甲酸裂解酶因而无法在厌氧条件下生长的大肠杆菌突变菌株。发现一个重组克隆表现出广泛特异性的依赖NAD⁺的D-2-羟基酸脱氢酶活性。对相应基因（命名为hdhD）进行亚克隆并测序。所编码酶的推导氨基酸序列表明这是一个由333个残基组成的蛋白质，与干酪乳杆菌的D-2-羟基异己酸（即2-羟基-4-甲基戊酸）脱氢酶（D-HO-HxoDH）以及其他几种细菌的其他依赖NAD⁺的D-乳酸脱氢酶（D-LDH）密切相关。hdhD基因在λ噬菌体PL启动子的控制下过表达，并且该酶通过两步法进行纯化。结果表明，德氏乳杆菌保加利亚亚种的这种酶与干酪乳杆菌的酶一样，对除具有支链β-碳的底物之外的多种2-氧代酸底物都有活性。
