Devine Ellenor, Holmqvist Marie, Stensjö Karin, Lindblad Peter
Department of Photochemistry and Molecular Science, The Angström Laboratories, Uppsala University, Uppsala, Sweden.
BMC Microbiol. 2009 Mar 11;9:53. doi: 10.1186/1471-2180-9-53.
The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively.
In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase.
Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co-evolution could be the result of a close interaction between the protease and the large subunit of the [NiFe]-hydrogenases, a theory supported by protein-protein docking experiments performed with 3D-models. Finally we present data that may explain the specificity seen among hydrogenase specific proteases, the so called "HOXBOX"; an amino acid sequence specific for proteases of Hox-type. This opens the door for more detailed studies of the specificity found among hydrogenase specific proteases and the structural properties behind it.
[NiFe]氢化酶大亚基成熟过程的最后一步是由氢化酶特异性蛋白酶对C末端进行蛋白水解切割。与其他辅助蛋白不同，这些氢化酶蛋白酶被认为具有特异性，即一种类型的氢化酶特异性蛋白酶只切割一种类型的氢化酶。在蓝细菌中，这是通过分别对摄取型或双向氢化酶特异的hupW或hoxW的基因产物实现的。丝状蓝细菌点状念珠藻ATCC 29133和念珠藻属菌株PCC 7120可能分别含有单一的摄取型氢化酶或同时含有摄取型和双向氢化酶。
为了研究蓝细菌中的这些蛋白酶，对点状念珠藻ATCC 29133中的hupW以及念珠藻属菌株PCC 7120中的hupW和hoxW进行了转录分析。这些研究揭示了众多转录起始点以及NtcA（hupW）和LexA（hoxW）的假定结合位点。为了研究氢化酶特异性蛋白酶之间的多样性和特异性，我们构建了一个系统发育树，该树揭示了几个亚组，这些亚组与先前描述的[NiFe]氢化酶的亚组有显著相似性。此外，还通过氨基酸序列分析以及与源自生物信息学研究的三维模型进行的蛋白质-蛋白质对接实验来探讨蛋白酶的特异性。这些研究揭示了一种所谓的“HOXBOX”；一种对Hox型蛋白酶特异的氨基酸序列，可能参与与氢化酶大亚基的对接。
我们的研究结果表明，氢化酶特异性蛋白酶与其切割的氢化酶受到相似的调控。系统发育研究的结果还表明，氢化酶和蛋白酶自古以来就共同进化，并且表明至少发生了一次主要的水平基因转移。这种共同进化可能是蛋白酶与[NiFe]氢化酶大亚基之间密切相互作用的结果，这一理论得到了用三维模型进行的蛋白质-蛋白质对接实验的支持。最后，我们提供的数据可能解释了氢化酶特异性蛋白酶之间所观察到的特异性，即所谓的“HOXBOX”；一种对Hox型蛋白酶特异的氨基酸序列。这为更详细地研究氢化酶特异性蛋白酶之间的特异性及其背后的结构特性打开了大门。
