Schmitz O, Boison G, Hilscher R, Hundeshagen B, Zimmer W, Lottspeich F, Bothe H
Botanisches Institut, Universität zu Köln, Germany.
Eur J Biochem. 1995 Oct 1;233(1):266-76. doi: 10.1111/j.1432-1033.1995.266_1.x.
An 8.9-kb segment with hydrogenase genes from the cyanobacterium Anabaena variabilis has been cloned and sequenced. The sequences show homology to the methyl-viologen-reducing hydrogenases from archaebacteria and, even more striking, to the NAD(+)-reducing enzymes from Alcaligenes eutrophus and Nocardia opaca as well as to the NADP(+)-dependent protein from Desulfovibrio fructosovorans. The cluster from A. variabilis contains genes coding for both the hydrogenase heterodimer (hoxH and hoxY) and for the diaphorase moiety (hoxU and hoxF) described for the A. eutrophus enzyme. In A. variabilis the gene cluster is split by two open reading frames (between hoxY and hoxH and between hoxU and hoxY, respectively), and a probably non-coding 0.9-kb segment in an unusual way. The hoxH partial sequence from Anabaena 7119 and Anacystis nidulans was amplified by PCR. Using the labeled segment from A. 7119 as probe, Southern analysis revealed homologous gene segments in the cyanobacteria A. 7119, Anabaena cylindrica, Anacystis nidulans and A. variabilis. The bidirectional hydrogenase from A. nidulans was purified and digests were sequenced. The amino acid sequences obtained showed partial identities to the amino acid sequences deduced from the DNA data of the 8.9-kb segment from A. variabilis. Therefore the 8.9-kb segment contains the genes coding for the bidirectional, reversible hydrogenase from cyanobacteria. Crude extracts from A. nidulans perform NAD(P)H-dependent H2 evolution corroborating the molecular biological demonstration of the NAD(P)(+)-dependent hydrogenase in cyanobacteria.
已克隆并测序了来自多变鱼腥蓝细菌的一段含有氢化酶基因的8.9 kb片段。这些序列与古细菌中还原甲基紫精的氢化酶具有同源性，更引人注目的是，与产碱杆菌和不透明诺卡氏菌中还原NAD(+)的酶以及与果糖脱硫弧菌中依赖NADP(+) 的蛋白具有同源性。来自多变鱼腥蓝细菌的基因簇包含编码氢化酶异二聚体（hoxH和hoxY）以及产碱杆菌酶中所述的双氢酶部分（hoxU和hoxF）的基因。在多变鱼腥蓝细菌中，基因簇被两个开放阅读框分别在hoxY和hoxH之间以及hoxU和hoxY之间以一种不寻常的方式隔开，还有一个可能是非编码的0.9 kb片段。通过PCR扩增了来自鱼腥蓝细菌7119和集胞藻的hoxH部分序列。使用来自鱼腥蓝细菌7119的标记片段作为探针，Southern分析揭示了在鱼腥蓝细菌7119、圆柱鱼腥蓝细菌、集胞藻和多变鱼腥蓝细菌中存在同源基因片段。纯化了来自集胞藻的双向氢化酶并对其消化产物进行了测序。获得的氨基酸序列与从多变鱼腥蓝细菌8.9 kb片段的DNA数据推导的氨基酸序列具有部分同一性。因此，该8.9 kb片段包含编码蓝细菌双向、可逆氢化酶的基因。来自集胞藻的粗提取物可进行依赖NAD(P)H的H2释放，这证实了蓝细菌中依赖NAD(P)(+)的氢化酶的分子生物学证明。
