Rákhely Gábor, Kovács Akos T, Maróti Gergely, Fodor Barna D, Csanádi Gyula, Latinovics Dóra, Kovács Kornél L
Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, University of Szeged, H-6726 Szeged, Hungary.
Appl Environ Microbiol. 2004 Feb;70(2):722-8. doi: 10.1128/AEM.70.2.722-728.2004.
Structural genes coding for two membrane-associated NiFe hydrogenases in the phototrophic purple sulfur bacterium Thiocapsa roseopersicina (hupSL and hynSL) have recently been isolated and characterized. Deletion of both hydrogenase structural genes did not eliminate hydrogenase activity in the cells, and considerable hydrogenase activity was detected in the soluble fraction. The enzyme responsible for this activity was partially purified, and the gene cluster coding for a cytoplasmic, NAD+-reducing NiFe hydrogenase was identified and sequenced. The deduced gene products exhibited the highest similarity to the corresponding subunits of the cyanobacterial bidirectional soluble hydrogenases (HoxEFUYH). The five genes were localized on a single transcript according to reverse transcription-PCR experiments. A sigma54-type promoter preceded the gene cluster, suggesting that there was inducible expression of the operon. The Hox hydrogenase was proven to function as a truly bidirectional hydrogenase; it produced H2 under nitrogenase-repressed conditions, and it recycled the hydrogen produced by the nitrogenase in cells fixing N2. In-frame deletion of the hoxE gene eliminated hydrogen evolution derived from the Hox enzyme in vivo, although it had no effect on the hydrogenase activity in vitro. This suggests that HoxE has a hydrogenase-related role; it likely participates in the electron transfer processes. This is the first example of the presence of a cyanobacterial-type, NAD+-reducing hydrogenase in a phototrophic bacterium that is not a cyanobacterium. The potential physiological implications are discussed.
编码光合紫色硫细菌玫瑰色硫囊菌中两种膜相关镍铁氢化酶的结构基因（hupSL和hynSL）最近已被分离和鉴定。删除这两个氢化酶结构基因并未消除细胞中的氢化酶活性，并且在可溶部分中检测到相当高的氢化酶活性。负责这种活性的酶被部分纯化，并且鉴定并测序了编码细胞质NAD +还原镍铁氢化酶的基因簇。推导的基因产物与蓝藻双向可溶性氢化酶（HoxEFUYH）的相应亚基具有最高的相似性。根据逆转录 - PCR实验，这五个基因位于单个转录本上。基因簇之前有一个sigma54型启动子，表明该操纵子存在诱导表达。已证明Hox氢化酶作为一种真正的双向氢化酶发挥作用；它在固氮酶抑制条件下产生H2，并在固定N2的细胞中循环利用固氮酶产生的氢气。hoxE基因的框内缺失消除了体内源自Hox酶的氢气释放，尽管它对体外氢化酶活性没有影响。这表明HoxE具有与氢化酶相关的作用；它可能参与电子传递过程。这是在非蓝藻的光合细菌中存在蓝藻型NAD +还原氢化酶的第一个例子。讨论了潜在的生理意义。
