CHARACTERIZATION OF AN OPERON ENCODING AN NADP-REDUCING HYDROGENASE IN DESULFOVIBRIO FRUCTOSOVORANS
CHARACTERIZATION OF AN OPERON ENCODING AN NADP-REDUCING HYDROGENASE IN DESULFOVIBRIO FRUCTOSOVORANS
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DOI：
10.1128/jb.177.10.2628-2636.1995
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发表时间：
1995-05-01
影响因子：
3.2
通讯作者：
BELAICH, JP
中科院分区：
文献类型：
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作者：
MALKI, S;SAIMMAIME, I;BELAICH, JP
克隆并测序了来自果糖脱硫弧菌的基因组 DNA 片段，该片段与普通脱硫弧菌 Hildenborough 的 hydAB 基因强杂交。发现该片段包含四个基因，名为 hnd4、hndB、hndC 和 hndD。序列同源性分析表明，HndA 与 Bos taunts 复合物 I 的 24 kDa 亚基、脱氮副球菌 NADH 脱氢酶 I 型的 25 kDa 亚基以及真养产碱菌 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未表现出任何显着的同源性。 HndC 与 HoxF 的 C 端结构域和 B. taurus 复合体 I 的 51 kDa 亚基分别显示 37% 和 33% 的同一性，并且具有能够结合 1 个黄素单核苷酸、1 个 NAD 和 3 个 [4Fe-4S] 簇所需的结构特征。 HndD 与来自巴氏梭菌的氢化酶 I 以及来自 D. vulgaris Hildenborough 的 HydC 和 HydA 分别具有 40%、42% 和 48% 的同一性。在 D. fructosovorans 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存在于同一转录单位上。通过使用T7启动子/聚合酶系统在大肠杆菌中进行hndABCD的异源表达来测量四种多肽的大小。 hndA、hndB、hndC 和 hndD 的产物分别为 18.8、13.8、52 和 63.4 kDa。一种 hndC 缺失突变体，称为 SM3，是通过进行标记交换诱变构建的。使用针对 HndC 的抗体对 D. fructosovorans 野生型和 SM3 菌株的细胞提取物进行免疫印迹研究，表明仅在野生型菌株的提取物中识别出 52-kDa 蛋白质。在 D. fructosovorans 野生型的可溶性提取物中，当 H. 存在时，观察到 NADP 减少了 10 倍，但从未发生过 H-2 依赖性 NAD 减少。这种 H-2 依赖性 NADP 还原酶活性在 SM3 提取物中完全消失。这些结果表明，hnd 操纵子实际上编码 D. fructosovorans 中的 NADP 还原氢化酶。
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 hnd4, 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 taunts 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. 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.