Porthun Antje, Bernhard Michael, Friedrich Bärbel
Institut für Biologie, Humboldt-Universität zu Berlin, Chausseestr. 117, 10115 Berlin, Germany.
Arch Microbiol. 2002 Feb;177(2):159-66. doi: 10.1007/s00203-001-0371-5. Epub 2001 Nov 20.
The actinomycete Rhodococcus opacus MR11 harbors a bidirectional NAD-reducing [NiFe] hydrogenase (SH). This cytoplasmic enzyme is composed of two heterodimeric modules which catalyze distinct enzymatic activities. The hydrogenase moiety mediates H(2):benzyl viologen oxidoreductase activity and the FMN-containing diaphorase module displays NADH:benzyl viologen oxidoreductase activity. The SH of Rh. opacus resembles [NiFe] hydrogenases present in strains of the proteobacterium Ralstonia eutropha and in species of cyanobacteria. Heterologous expression of active [NiFe] hydrogenases failed in most cases due to protein-assisted maturation processes implicated in the assembly of the NiFe bimetallic site. This study reports on the construction of a recombinant plasmid harboring the four SH subunit genes hoxFUYH and the associated endopeptidase gene hoxW from Rh. opacus under the regime of the SH promoter from R. eutropha H16. The resulting recombinant plasmid restored lithoautotrophic growth in a R. eutropha mutant impaired in H(2)-oxidizing ability. The SH of Rh. opacus was functionally active in R. eutropha and displayed the typical features described for its natural host. It readily dissociated in vitro into two active subforms. Dissociation was accompanied by the loss of the H(2)-dependent NAD-reducing activity, which was partially reconstituted by addition of 5 mM MgSO(4) and 0.5 mM NiCl(2). Activity and stability of the SH from Rh. opacus were enhanced almost three-fold by co-overexpression of the SH-associated metal insertion genes hypA2B2F2 of R. eutropha. Under optimal conditions the heterologously expressed Rh. opacus SH catalyzed NAD-reduction at a specific activity of 1.7 units per mg protein, which is approximately 30% of the yield obtained for the R. eutropha SH. The results indicate that, despite an enormous phylogenetic distance of the two bacterial species, their SH proteins are highly related.
放线菌迟钝红球菌MR11含有一种双向NAD还原型[NiFe]氢化酶（SH）。这种胞质酶由两个异源二聚体模块组成，它们催化不同的酶活性。氢化酶部分介导H₂：苄基紫精氧化还原酶活性，含FMN的递氢酶模块表现出NADH：苄基紫精氧化还原酶活性。迟钝红球菌的SH类似于存在于真养产碱杆菌菌株和蓝细菌物种中的[NiFe]氢化酶。在大多数情况下，活性[NiFe]氢化酶的异源表达失败，这是由于蛋白质辅助成熟过程与NiFe双金属位点的组装有关。本研究报道了构建一个重组质粒，该质粒含有来自迟钝红球菌的四个SH亚基基因hoxFUYH和相关的内肽酶基因hoxW，其受真养产碱杆菌H16的SH启动子调控。所得重组质粒恢复了真养产碱杆菌一个H₂氧化能力受损的突变体的自养生长。迟钝红球菌的SH在真养产碱杆菌中具有功能活性，并表现出其天然宿主所描述的典型特征。它在体外很容易解离成两种活性亚形式。解离伴随着H₂依赖性NAD还原活性的丧失，通过添加5 mM MgSO₄和0.5 mM NiCl₂可部分恢复该活性。通过真养产碱杆菌的SH相关金属插入基因hypA2B2F2的共过表达，迟钝红球菌SH的活性和稳定性提高了近三倍。在最佳条件下，异源表达的迟钝红球菌SH催化NAD还原，比活性为每毫克蛋白质1.7单位，约为真养产碱杆菌SH产量的30%。结果表明，尽管这两种细菌在系统发育上距离很远，但它们的SH蛋白高度相关。
