NAD+-DEPENDENT D-2-HYDROXYISOCAPROATE DEHYDROGENASE OF LACTOBACILLUS-DELBRUECKII SUBSP BULGARICUS - GENE CLONING AND ENZYME CHARACTERIZATION
NAD+-DEPENDENT D-2-HYDROXYISOCAPROATE DEHYDROGENASE OF LACTOBACILLUS-DELBRUECKII SUBSP BULGARICUS - GENE CLONING AND ENZYME CHARACTERIZATION
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DOI：
10.1111/j.1432-1033.1994.00439.x
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发表时间：
1994-09-01
期刊：
影响因子：
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通讯作者：
DELCOUR, J
中科院分区：
文献类型：
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作者：
BERNARD, N;JOHNSEN, K;DELCOUR, J
来自德氏乳杆菌亚种的基因组文库。保加利亚菌株被用来补充缺乏乳酸脱氢酶和丙酮酸甲酸裂合酶的大肠杆菌突变菌株，因此无法厌氧生长。发现一种重组克隆表现出广泛的特异性 NAD(+) 依赖性 D-2-羟酸脱氢酶活性。相应的基因（命名为 hdhD）被亚克隆并测序。编码酶的推导氨基酸序列表明，该蛋白具有 333 个残基，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) 和来自其他几种细菌物种的其他 NAD(+) 依赖性 D-乳酸脱氢酶 (D-LDH) 密切相关。 hdhD 基因在 lambda 噬菌体 P-L 启动子的控制下过表达，并用两步法纯化酶。德氏乳杆菌亚种。保加利亚酶与干酪乳杆菌酶一样，对多种 2-含氧酸底物具有活性，但具有支链 β-碳的底物除外。
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 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-oxoacid substrates except those having a branched beta-carbon.