Identification of essential catalytic residues of the cyclase NisC involved in the biosynthesis of nisin
Identification of essential catalytic residues of the cyclase NisC involved in the biosynthesis of nisin
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DOI：
10.1074/jbc.m701802200
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发表时间：
2007-07-20
影响因子：
4.8
通讯作者：
van der Donk, Wilfred A.
中科院分区：
文献类型：
--
作者：
Li, Bo;van der Donk, Wilfred A.
Nisin是一种翻译后修饰的抗菌肽，在食品工业中广泛应用了几十年。它包含五个不同大小的环硫醚交联，由一种酶（NisC）安装，这种酶催化半胱氨酸向脱氢氨基酸的添加。NisC最近的x射线晶体结构首次提供了对nisin生物合成过程中负责环化步骤的催化残基的见解。在本研究中，保守残基His（212）、Arg（280）、Asp（141）和Tyr（285）以及活性位点锌的配体Cys（284）、Cys（330）和His（331）被定点突变取代。结合研究表明，所有突变体对NisA具有相似的亲和力。活性测定表明，His（212）和Asp（141）对最终产物的正确环化是必需的，而Arg（280）和Tyr（285）则不是。锌配体突变为丙氨酸也使酶活性丧失，这些突变蛋白中锌含量降低。这些结果表明，锌对活性是必不可少的，并支持锌用于激活底物中的半胱氨酸进行亲核攻击的模型。这些发现也反驳了Arg（280）和Tyr（285）作为一般酸/碱的活性位点在环化机制中的重要作用。
Nisin is a post-translationally modified antimicrobial peptide that has been widely used in the food industry for several decades. It contains five cyclic thioether cross-links of varying sizes that are installed by a single enzyme, NisC, that catalyzes the addition of cysteines to dehydroamino acids. The recent x-ray crystal structure of NisC has provided the first insights into the catalytic residues responsible for the cyclization step during nisin biosynthesis. In this study, the conserved residues His(212), Arg(280), Asp(141), and Tyr(285) as well as the ligands to the zinc in the active site (Cys(284), Cys(330), and His(331)) were substituted by site-directed mutagenesis. Binding studies showed that all mutants had similar affinities for NisA. Activity assays showed that whereas His(212) and Asp(141) were essential for correct cyclization as judged by the antimicrobial activity of the final product, Arg(280) and Tyr(285) were not. Mutation of zinc ligands to alanine also abolished the enzymatic activity, and these mutant proteins were shown to contain decreased levels of zinc. These results show that the zinc is essential for activity and support a model in which the zinc is used to activate the cysteines in the substrate for nucleophilic attack. These findings also argue against an essential role of Arg(280) and Tyr(285) as an active site general acid/base in the mechanism of cyclization.