Ma Hongchu, Gao Yong, Zhao Fangyuan, Zhong Jin
Wei Sheng Wu Xue Bao. 2015 Jan 4;55(1):50-8.
To reconstitute the in vitro catalytic activity of the individual dehydratase or cyclase domain of bifunctional bovicin HJ50 synthase BovM, and lay a foundation for the further investigation of catalytic mechanism of class II lantibiotic synthase LanM.
The truncated proteins of BovM containing the N-terminal dehydratase domain or C-terminal cyclase domain were expressed in E. coli and purified. Substrate BovA, the precursor of bovicin HJ50, was incubated with these truncated BovM proteins in in vitro reaction system. The antimicrobial activity assay and MALDI-TOF MS analysis were used to monitor the dehydratase or cyclase activity of these truncated proteins. Meanwhile, the synergistic activities of both truncated proteins were tested in vivo and in vitro.
The N- and C-terminal domains of BovM possessed dehydration and cyclization activity respectively. However, no synergistic activity was detected between these two functional domains.
The individual functional domains of BovM could execute their corresponding functions independently, but the intactness of BovM was important for its full modification activity.
重构双功能博维菌素HJ50合酶BovM的单个脱水酶或环化酶结构域的体外催化活性，为进一步研究II类羊毛硫抗生素合酶LanM的催化机制奠定基础。
在大肠杆菌中表达并纯化含有N端脱水酶结构域或C端环化酶结构域的BovM截短蛋白。将博维菌素HJ50的前体底物BovA与这些截短的BovM蛋白在体外反应体系中孵育。采用抗菌活性测定和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析来监测这些截短蛋白的脱水酶或环化酶活性。同时，在体内和体外测试这两种截短蛋白的协同活性。
BovM的N端和C端结构域分别具有脱水和环化活性。然而，在这两个功能结构域之间未检测到协同活性。
BovM的单个功能结构域能够独立执行其相应功能，但BovM的完整性对其完全修饰活性很重要。
