In vitro activity of the nisin dehydratase NisB
In vitro activity of the nisin dehydratase NisB
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DOI：
10.1073/pnas.1222488110
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发表时间：
2013-04-30
影响因子：
11.1
通讯作者：
van der Donk, Wilfred A.
中科院分区：
文献类型：
--
作者：
Garg, Neha;Salazar-Ocampo, Luis M. A.;van der Donk, Wilfred A.
几类核糖体合成和后修饰肽的生物合成涉及丝氨酸和苏氨酸残基的脱水。对于I类羊毛硫抗生素、硫肽和goadsporin，这种脱水由羊毛硫菌素生物合成酶B（LanB）或LanB样蛋白催化。尽管自1992年以来已经研究了LanB蛋白，但其脱水活性的体外重建一直是难以捉摸的。我们在这里展示了参与食品防腐剂乳链菌肽（NisB）生物合成的酶的体外活性。在体外，NisB脱水其底物肽NisA八倍的存在下，谷氨酸，ATP，Mg 2+，和细菌细胞提取物的核糖体/膜部分。NisB的23个高度保守的残基的突变鉴定了脱水活性所必需的许多氨基酸。此外，这些诱变研究鉴定了三种突变体R786A、R826A和H961A，其导致NisA底物的多重谷氨酰化。在大肠杆菌共表达NisA与这些突变体和体外试验期间观察到谷氨酸化。用WT NisB处理谷氨酰化底物导致脱水NisA，表明谷氨酰化肽是脱水的中间体。总的来说，这些研究表明，脱水涉及丝氨酸和苏氨酸侧链的谷氨酰化，然后消除。后一步骤在维吉尼亚霉素抗性蛋白维吉尼亚霉素B裂解酶中具有先例。这些研究将有助于调查其他LanB蛋白参与羊毛硫抗生素，硫肽，和goadsporin的生物合成。
The biosynthesis of several classes of ribosomally synthesized and posttranslationally modified peptides involves dehydration of serine and threonine residues. For class I lantibiotics, thiopeptides, and goadsporin, this dehydration is catalyzed by lanthionine biosynthetic enzyme B (LanB) or LanB-like proteins. Although LanB proteins have been studied since 1992, in vitro reconstitution of their dehydration activity has been elusive. We show here the in vitro activity of the dehydratase involved in the biosynthesis of the food preservative nisin (NisB). In vitro, NisB dehydrated its substrate peptide NisA eight times in the presence of glutamate, ATP, Mg2+, and the ribosomal/membrane fraction of bacterial cell extract. Mutation of 23 highly conserved residues of NisB identified a number of amino acids that are essential for dehydration activity. In addition, these mutagenesis studies identified three mutants, R786A, R826A, and H961A, that result in multiple glutamylations of the NisA substrate. Glutamylation was observed during both Escherichia coli coexpression of NisA with these mutants and in vitro assays. Treatment of the glutamylated substrate with WT NisB results in dehydrated NisA, suggesting that the glutamylated peptide is an intermediate in dehydration. Collectively, these studies suggest that dehydration involves glutamylation of the side chains of Ser and Thr followed by elimination. The latter step has precedent in the virginiamycin resistance protein virginiamycin B lyase. These studies will facilitate investigation of other LanB proteins involved in the biosynthesis of lantibiotics, thiopeptides, and goadsporin.