Malki S, Saimmaime I, De Luca G, Rousset M, Dermoun Z, Belaich J P
Centre National de la Recherche Scientifique, Marseilles, France.
J Bacteriol. 1995 May;177(10):2628-36. doi: 10.1128/jb.177.10.2628-2636.1995.
A genomic DNA fragment from Desulfovibrio fructosovorans, which strongly hybridized with the hydAB genes from Desulfovibrio vulgaris Hildenborough, was cloned and sequenced. This fragment was found to contain four genes, named hndA, hndB, hndC, and hndD. Analysis of the sequence homologies indicated that HndA shows 29, 21, and 26% identity with the 24-kDa subunit from Bos taurus complex I, the 25-kDa subunit from Paracoccus denitrificans NADH dehydrogenase type I, and the N-terminal domain of HoxF subunit of the NAD-reducing hydrogenase from Alcaligenes eutrophus, respectively. HndB does not show any significant homology with any known protein. HndC shows 37 and 33% identity with the C-terminal domain of HoxF and the 51-kDa subunit from B. taurus complex I, respectively, and has the requisite structural features to be able to bind one flavin mononucleotide, one NAD, and three [4Fe-4S] clusters. HndD has 40, 42, and 48% identity with hydrogenase I from Clostridium pasteurianum and HydC and HydA from D. vulgaris Hildenborough, respectively. The 4.5-kb length of the transcripts expressed in D. fructosovorans and in Escherichia coli (pSS13) indicated that all four genes were present on the same transcription unit. The sizes of the four polypeptides were measured by performing heterologous expression of hndABCD in E. coli, using the T7 promoter/polymerase system. The products of hndA, hndB, hndC, and hndD were 18.8, 13.8, 52, and 63.4 kDa, respectively. One hndC deletion mutant, called SM3, was constructed by performing marker exchange mutagenesis. Immunoblotting studies carried out on cell extracts from D. fructosovorans wild-type and SM3 strains, using antibodies directed against HndC, indicated that the 52-kDa protein was recognized in extracts from the wild-type strain only. In soluble extracts from D. fructosovorans wild type, a 10-fold induction of NADP reduction was observed when H(2) was present, but no H(2)-dependent NAD reduction ever occurred. This H(2)-dependent NADP reductase activity disappeared completely in extracts from SM3. These results indicate that the hnd operon actually encodes an NAdP-reducing hydrogenase in D. fructosovorans.
从果糖脱硫弧菌中克隆并测序了一个基因组DNA片段，该片段与普通脱硫弧菌希登伯勒菌株的hydAB基因强烈杂交。发现该片段包含四个基因，命名为hndA、hndB、hndC和hndD。序列同源性分析表明，HndA与牛复合体I的24 kDa亚基、反硝化副球菌I型NADH脱氢酶的25 kDa亚基以及嗜碱产碱菌NAD还原氢化酶的HoxF亚基的N端结构域分别具有29%、21%和26%的同一性。HndB与任何已知蛋白质均无明显同源性。HndC与HoxF的C端结构域以及牛复合体I的51 kDa亚基分别具有37%和33%的同一性，并且具有能够结合一个黄素单核苷酸、一个NAD和三个[4Fe-4S]簇的必要结构特征。HndD与巴氏梭菌的氢化酶I以及普通脱硫弧菌希登伯勒菌株的HydC和HydA分别具有40%、42%和48%的同一性。在果糖脱硫弧菌和大肠杆菌（pSS13）中表达的转录本长度为4.5 kb，表明所有四个基因都位于同一个转录单元上。通过使用T7启动子/聚合酶系统在大肠杆菌中对hndABCD进行异源表达，测定了这四种多肽的大小。hndA、hndB、hndC和hndD的产物分别为18.8 kDa、13.8 kDa、52 kDa和63.4 kDa。通过进行标记交换诱变构建了一个hndC缺失突变体，称为SM3。使用针对HndC的抗体对果糖脱硫弧菌野生型和SM3菌株的细胞提取物进行免疫印迹研究，结果表明仅在野生型菌株的提取物中识别出52 kDa的蛋白质。在果糖脱硫弧菌野生型的可溶性提取物中，当存在H₂时观察到NADP还原有10倍的诱导，但从未发生过依赖H₂的NAD还原。这种依赖H₂的NADP还原酶活性在SM3的提取物中完全消失。这些结果表明，hnd操纵子实际上在果糖脱硫弧菌中编码一种NADP还原氢化酶。
