前导肽切割位点对Ⅱ类羊毛硫细菌素切割酶活性的影响

Effects of cleavage sites of leader peptides on the activities of class Ⅱ lantibiotic peptidases

摘要:

【背景】Ⅱ类羊毛硫细菌素大多是由革兰氏阳性菌的核糖体合成并经过翻译后修饰产生的小肽，其生物合成的最后一步是由转运蛋白LanT N端的肽酶结构域对前导肽进行切割，释放出有活性的羊毛硫细菌素，但目前关于该类羊毛硫细菌素前导肽的切割机制尚不清楚。【目的】考察前导肽切割位点对不同链球菌来源的肽酶结构域BovT150和SboT150酶切活性的影响。【方法】运用不依赖连接酶的定点突变技术构建前导肽切割位点突变的前体蛋白表达载体，在大肠杆菌(Escherichia coli)中分别表达纯化野生型前体(BovAm和SboAm)、突变型前体及对应的切割酶(BovT150和SboT150)，构建体外酶切体系，利用HPLC、抑菌活性分析和MALDI-TOF MS检测前导肽的切除情况。【结果】BovT150不仅能够切割BovAm的GG和GA位点，也能切割SboAm的GG和GA位点，并且对切割位点为Gly的前体切割活性较高；SboT150仅能切割SboAm的GG和GA位点，而对切割位点为Ala的活性较高。【结论】II类羊毛硫细菌素前导肽切割位点氨基酸残基的改变不同程度地影响切割酶的切割效率。

Abstract:

[Background] Class Ⅱ lantibiotics are ribosomally synthesized and post-translationally modified peptides, mainly produced by Gram-positive bacteria. In the last step of its biosynthesis, the N-terminal peptidase domain of transporter protein LanT cleaves the leader peptide to produce active lantibiotics. However, the removal mechanism of leader peptide in this class lantibiotics is still not clear. [Objective] To investigate the effects of cleavage sites on the activity of peptidases domain BovT150 and SboT150 from different streptococci. [Methods] Expression vectors for the precursor peptides with mutated cleavage sites were constructed by site-directed ligase-independent mutagenesis and then the wild-type precursors (BovAm and SboAm), their mutant precursors, as well as the corresponding peptidases (BovT150 and SboT150) were expressed and purified in E. coli. The precursors were separately incubated with each peptidase in vitro and the removal efficiencies of the leader peptides were assessed by HPLC, antimicrobial activity assay and MALDI-TOF MS. [Results] Both cleavage sites GG and GA of BovAm and SboAm allowed BovT150 to retain peptidase activity, and Gly was more suitable to be processed by BovT150. Only cleavage sites GG and GA of SboAm were accessible to SboT150, which cleaved Ala more efficiently. [Conclusion] The change of amino acid residues at the cleavage sites of leader peptides affected the efficiencies of class Ⅱ lantibiotic peptidases in varying degrees.