Characterization of Leader Peptide Binding During Catalysis by the Nisin Dehydratase NisB,Journal of the American Chemical Society

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Characterization of Leader Peptide Binding During Catalysis by the Nisin Dehydratase NisB
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2018-03-14 , DOI: 10.1021/jacs.7b13506
Lindsay M. Repka ₁ , Kenton J. Hetrick ₁ , See Hyun Chee ₁ , Wilfred A. van der Donk ₁

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The dehydratase NisB performs stepwise tRNAGlu-dependent glutamylation of Ser/Thr residues and subsequent glutamate elimination to effect eight dehydrations in the biosynthesis of the antibacterial peptide nisin. Its substrate, NisA, bears a C-terminal core peptide that is modified and an N-terminal leader peptide (LP) that is not modified but that is required for efficient dehydration. To elucidate the mechanism of LP-NisB interactions during dehydration, we engineered a disulfide that covalently links the NisA LP to NisB. The enzyme fully dehydrated tethered NisA, confirming the functional LP binding site and supporting a mechanism where NisB uses a single LP binding site for glutamylation and elimination. We also show an order of NisA and tRNAGlu binding to NisB that enables dehydration.

中文翻译：

Nisin 脱水酶 NisB 催化过程中前导肽结合的表征

脱水酶 NisB 执行 Ser/Thr 残基的逐步 tRNAGlu 依赖性谷氨酰化和随后的谷氨酸消除，以在抗菌肽乳酸链球菌肽的生物合成中实现八次脱水。它的底物 NisA 带有一个经过修饰的 C 端核心肽和一个未经修饰但有效脱水所需的 N 端前导肽 (LP)。为了阐明脱水过程中 LP-NisB 相互作用的机制，我们设计了一种二硫化物，将 NisA LP 与 NisB 共价连接。该酶完全脱水束缚 NisA，确认功能性 LP 结合位点并支持 NisB 使用单个 LP 结合位点进行谷氨酰化和消除的机制。我们还展示了 NisA 和 tRNAGlu 与 NisB 结合的顺序，从而使脱水成为可能。

更新日期：2018-03-14

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