An unusual subtilisin-like serine protease is essential for biogenesis of quinohemoprotein amine dehydrogenase
An unusual subtilisin-like serine protease is essential for biogenesis of quinohemoprotein amine dehydrogenase
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一种不寻常的枯草杆菌蛋白酶样丝氨酸蛋白酶对于醌血红素蛋白胺脱氢酶的生物合成至关重要
DOI：
10.1074/jbc.m111.324756
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Toshihide Okajima.
中科院分区：
文献类型：
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作者：
Tadashi Nakai;Kazutoshi Ono;Shun'ichi Kuroda;Katsuyuki Tanizawa;Toshihide Okajima.
奎诺血红素蛋白胺脱氢酶（Quinohemoprotein amine dehydrogenase，QHNDH）是一种存在于革兰氏阴性菌周质中的αβγ异源三聚体，催化正丁胺等脂肪胺氧化脱氨，并将其同化为碳源和能源。成熟QHNDH的γ亚基含有蛋白质衍生的醌辅因子、半胱氨酸醌和Cys与Asp或Glu残基之间的三个肽内硫醚交联。在其细胞质新生形式中，γ亚基具有28个残基的N-末端前导肽，其对于活性QHNDH的产生是必需的，但必须在随后的成熟过程中去除。在此，我们描述了一种枯草杆菌蛋白酶样丝氨酸蛋白酶（ORF 5）在QHNDH生物合成中的作用，ORF 5的破坏导致细菌细胞在含有正丁胺的培养基中生长受到抑制，并产生无活性的QHNDH，其中γ亚基保留前导肽。质粒ORF 5的供应恢复了细胞生长和含有正确加工的γ亚基的活性QHNDH的产生。ORF 5在大肠杆菌中表达，但其催化三联体突变体不切割替代γ亚基前导肽的合成肽，尽管非常缓慢。切割的前导肽仍然不稳定地与ORF 5结合，最有可能作为连接到活性位点Ser残基的酰基酶中间体。这些结果表明ORF 5是QHNDH生物合成所必需的，作为加工蛋白酶几乎以一次性方式切割γ亚基前导肽。
Quinohemoprotein amine dehydrogenase (QHNDH), an αβγ heterotrimer present in the periplasm of several Gram-negative bacteria, catalyzes the oxidative deamination of various aliphatic amines such asn-butylamine for assimilation as carbon and energy sources. The γ subunit of mature QHNDH contains a protein-derived quinone cofactor, cysteine tryptophylquinone, and three intrapeptidyl thioether cross-links between Cys and Asp or Glu residues. In its cytoplasmic nascent form, the γ subunit has a 28-residue N-terminal leader peptide that is necessary for the production of active QHNDH but must be removed in the following maturation process. Here, we describe the role of a subtilisin-like serine protease encoded in the fifth ORF of then-butylamine-utilizing operon ofParacoccus denitrificans(termed ORF5) in QHNDH biogenesis.ORF5disruption caused bacterial cell growth inhibition inn-butylamine-containing medium and production of inactive QHNDH, in which the γ subunit retained the leader peptide. Supply of plasmid-encodedORF5restored the cell growth and production of active QHNDH, containing the correctly processed γ subunit. ORF5 expressed inEscherichia colibut not its catalytic triad mutant cleaved synthetic peptides surrogating for the γ subunit leader peptide, although extremely slowly. The cleaved leader peptide remained unstably bound to ORF5, most likely as an acyl enzyme intermediate attached to the active-site Ser residue. These results demonstrate that ORF5 is essential for QHNDH biogenesis, serving as a processing protease to cleave the γ subunit leader peptide nearly in a disposable manner.