Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit--identification of a soluble precursor of the small subunit in a hypB mutant.
Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit--identification of a soluble precursor of the small subunit in a hypB mutant.
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编码大肠杆菌氢化酶 2 小亚基的基因的重新分配——鉴定 hypB 突变体中小亚基的可溶性前体。
DOI：
10.1046/j.1432-1327.1998.2550746.x
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
D. Boxer
中科院分区：
文献类型：
--
作者：
F. Sargent;Stuart P. Ballantine;P. Rugman;Tracy Palmer;D. Boxer
已从细胞质膜的周质面分离出来自大肠杆菌的氢化酶2的活性胰蛋白酶片段，并对大亚基和小亚基进行N-末端测序。大亚基由hybC基因编码，除了在其生物合成过程中去除起始甲硫氨酸外，没有显示N-末端加工。N-末端和随后的内部胰蛋白酶片段氨基酸序列都表明，小亚基既不是由hybA编码的，hybA是先前鉴定为编码小亚基的基因[Menon等人（1994）J. Bacteriol. 176，4416-4423]，也没有hyb操纵子中的任何剩余基因。基因组序列分析显示存在一个开放的阅读框架，它可能编码蛋白水解的小亚基的肽序列。该基因被命名为hyb 0，位于hybA基因的直接上游，并与hybA基因的起点相隔两个核苷酸。Hyb 0与其他氢化酶小亚基氨基酸序列具有约40%的同一性，其合成具有N-末端信号序列，该信号序列含有可能需要用于酶输出的双精氨酸基序。在成熟酶中，小亚基在Ala 37之后被蛋白水解裂解。使用氢化酶2特异性抗体对过度产生重组Hyb 0或Hyb A的菌株进行免疫学分析，很容易将Hyb 0鉴定为小亚基。在一个多效性的hypB突变体，这是无法插入镍到活性位点，无论是大的和小的亚基积累作为未加工的，可溶性的形式，与两个亚基组装和处理在生物合成过程中以协调的方式一致。
An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced. The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N-terminal and the subsequent internal tryptic-fragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416-4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.