Laboratory of Molecular Microbiological Science, Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure, Hiroshima, Japan.
Microbiol Immunol. 2009 Dec;53(12):647-57. doi: 10.1111/j.1348-0421.2009.00175.x.
ASP is the only bacterial protease in the kexin group of the subtilisin family. Previous studies have revealed that the ORF2 protein encoded at the 3' end of the asp operon is required for ASP to change from a nascent form into an active form in the periplasm. However, the mechanism by which ORF2 makes contact and interacts with ASP in the maturation process remains unknown. The present study examined the effect of mutations in the carboxy-terminal region of ASP on the ASP maturation process. Both deletion-mutation and amino acid-substitution studies have demonstrated that the histidine residue at position 595 (His-595), the sixth residue from the carboxyl terminus of ASP, is highly involved in the generation of active ASP molecules. An analysis by pull-down assay revealed that mutation at His-595 reduces the efficacy of nascent ASP to transition into active ASP by reducing the ability of ASP to make contact and interact with ORF2. Thus, it appears likely that nascent ASP in the periplasm interacts with ORF2 via the carboxy-terminal region, and His-595 of ASP appears to be an indispensable residue in this interaction.
ASP 是枯草杆菌蛋白酶族中糜蛋白酶家族的唯一细菌蛋白酶。先前的研究表明，asp 操纵子 3' 末端编码的 ORF2 蛋白对于 ASP 在周质中从新生形式转变为活性形式是必需的。然而，ORF2 在成熟过程中与 ASP 接触和相互作用的机制尚不清楚。本研究探讨了 ASP 羧基末端区域突变对 ASP 成熟过程的影响。缺失突变和氨基酸取代研究均表明，ASP 羧基末端第六位的组氨酸残基 595（His-595）在生成活性 ASP 分子中起着重要作用。下拉分析表明，突变 His-595 会降低新生 ASP 向活性 ASP 转化的效率，降低 ASP 与 ORF2 接触和相互作用的能力。因此，周质中的新生 ASP 似乎通过羧基末端区域与 ORF2 相互作用，而 ASP 的 His-595 似乎是这种相互作用中不可或缺的残基。
