羊毛硫细菌素bovicin HJ50修饰酶BovM双功能域单独催化活性鉴定

Individual catalytic activity of two functional domains of bovicin HJ50 synthase BovM

摘要:

摘要:【目的】体外重建羊毛硫细菌素bovicin HJ50修饰酶BovM双功能域(脱水酶与环化酶功能域)各自的催化活性，为深入了解BovM催化机制奠定基础。【方法】在大肠杆菌(Escherichia coli)中分别异源表达纯化BovM含脱水功能域的N 端与含环化功能域的C端重组蛋白，构建体外反应体系，分别对其前肽底物BovA进行修饰，通过产物抑菌活性及MALDI-TOF MS分子量检测来鉴定两者的修饰活性;并通过体内体外两种方法检测双功能域蛋白之间的协同作用。【结果】BovM的N端脱水功能域及C端环化功能域分别具有脱 水与环化活性，但双功能域重组蛋白之间没有协同作用。【结论】BovM双功能域均可独立行使各自的催化功能，但BovM的完整结构对其正常的催化活性非常重要。

Abstract:

Abstract:［Objective］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．［Method］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．［Results］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．［Conclusion］The individual functional domains of BovM could execute their corresponding functions independently，but the intactness of BovM was important for its full modification activity.