Inokoshi Junji, Koyama Nobuhiro, Miyake Midori, Shimizu Yuji, Tomoda Hiroshi
Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
Sci Rep. 2016 Jul 26;6:30375. doi: 10.1038/srep30375.
Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient Rhodococcus jostii, and generated 36 lariatin variants of the precursor protein LarA to investigate the biosynthesis and the structure-activity relationships. The mutational analysis revealed that four amino acid residues (Gly1, Arg7, Glu8, and Trp9) in lariatin A are essential for the maturation and production in the biosynthetic machinery. Furthermore, the study on structure-activity relationships demonstrated that Tyr6, Gly11, and Asn14 are responsible for the anti-mycobacterial activity, and the residues at positions 15, 16 and 18 in lariatin A are critical for enhancing the activity. This study will not only provide a useful platform for genetically engineering Gram-positive bacterium-producing lasso peptides, but also an important foundation to rationally design more promising drug candidates for combatting tuberculosis.
拉里阿廷A是一种由五基因簇larABCDE编码的含18个残基的套索肽，具有强大的选择性抗分枝杆菌活性。其结构特征是一个N端大环内酰胺环，C端穿过该环形成刚性的套索原结结构。在本研究中，我们通过将携带larA突变基因的质粒转化到larA缺陷型约氏红球菌中的策略建立了一个汇聚表达系统，并产生了36种前体蛋白LarA的拉里阿廷变体，以研究其生物合成及构效关系。突变分析表明，拉里阿廷A中的四个氨基酸残基（Gly1、Arg7、Glu8和Trp9）对于生物合成机制中的成熟和产生至关重要。此外，构效关系研究表明，Tyr6、Gly11和Asn14负责抗分枝杆菌活性，拉里阿廷A中第15、16和18位的残基对于增强活性至关重要。本研究不仅将为基因工程改造产生套索肽的革兰氏阳性细菌提供一个有用的平台，也为合理设计更有前景的抗结核候选药物奠定重要基础。
