Dissecting the Maturation Steps of the Lasso Peptide Microcin J25 in vitro
Dissecting the Maturation Steps of the Lasso Peptide Microcin J25 in vitro
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DOI：
10.1002/cbic.201200016
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发表时间：
2012-05-07
期刊：
影响因子：
3.2
通讯作者：
Rebuffat, Sylvie
中科院分区：
文献类型：
--
作者：
Yan, Kok-Phen;Li, Yanyan;Rebuffat, Sylvie
Microcin J25是一类细菌核糖体多肽的原型，具有打结的拓扑结构(套索多肽)。它由一个八个残基的大内酰胺环组成，C-末端的尾部穿过这个环。它被生物合成为前体，由两种成熟酶(McjB/McjC)加工。先前已经通过在体内诱变前体多肽提供了对微霉素J25生物合成机制的见解。在这项研究中，我们首次在体外证明了McjB和McjC的不同功能，基于对反应中间产物的检测。McjB被鉴定为一种新的依赖于ATP的半胱氨酸蛋白酶，而McjC被证实为内酰胺合成酶。这两种酶在功能上是相互依赖的，很可能形成了结构复合体。利用突变的前体多肽直接研究它们的底物选择性。根据替换的不同，微囊素J25可以在体外产生具有套索或分支-环拓扑的变异体。
Microcin J25 is the archetype of a growing class of bacterial ribosomal peptides possessing a knotted topology (lasso peptides). It consists of an eight-residue macrolactam ring through which the C-terminal tail is threaded. It is biosynthesized as a precursor that is processed by two maturation enzymes (McjB/McjC). Insights into the mechanism of microcin J25 biosynthesis have been provided previously by mutagenesis of the precursor peptide in vivo. In this study we have demonstrated distinct functions of McjB and McjC in vitro for the first time, based on the detection of reaction intermediates. McjB was characterized as a new ATP-dependent cysteine protease, whereas McjC was confirmed to be a lactam synthetase. The two enzymes were functionally interdependent, likely forming a structural complex. Their substrate preference was directly investigated with the aid of mutated precursor peptides. Depending on the substitutions, microcin J25 variants with either a lasso or branched-cyclic topology could be generated in vitro.