Structural and Biochemical Characterization of AidC, a Quorum-Quenching Lactonase with Atypical Selectivity.
Structural and Biochemical Characterization of AidC, a Quorum-Quenching Lactonase with Atypical Selectivity.
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DOI：
10.1021/acs.biochem.5b00499
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发表时间：
2015-07-21
期刊：
影响因子：
2.9
通讯作者：
Fast W
中科院分区：
文献类型：
--
作者：
Mascarenhas R;Thomas PW;Wu CX;Nocek BP;Hoang QQ;Liu D;Fast W
群体猝灭催化剂具有潜在的应用前景，可以作为生物化学工具来研究细菌间的通讯途径，作为抗生物污垢的试剂，以及作为动植物的抗感染药物。本文对金黄色杆菌产生的N-酰基-L-高丝氨酸内切酶AIDC的结构和功能进行了研究。稳态动力学研究表明，添加锌的AIDC是迄今为止最有效的野生型群体猝灭酶之一，其对N-庚酰基-L-高丝氨酸内酯的kcat/Km值约为2×106M−1S−1。该酶具有更严格的底物选择性，Km值(首选底物约50μM)明显低于典型的AHLLactonase(约1 mm)。分别测定了AIDC及其产物N-己酰基-L-高丝氨酸的X-射线晶体结构。每个结构都以二聚体的形式存在，尺寸排斥层析结合多角度光散射也观察到了溶液中的二聚体齐聚。与以前描述的AHL内酯酶相比，这些结构显示了两个不典型的特征：一个是形成闭合结合口袋的α-螺旋，它提供了对AHL底物的亲和力并加强了选择性；另一个是活性部位His取代，通常在同源的磷酸二酯酶家族中发现。讨论了AHL内酯酶催化机理的意义。
Quorum-quenching catalysts are of interest for potential application as biochemical tools to interrogate interbacterial communication pathways, as anti-biofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-L-homoserine (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is one of the most efficient wild-type quorum-quenching enzymes characterized to date, with a kcat/KM value of approximately 2 × 106 M−1s−1 for N-heptanoyl-L-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 μM for preferred substrates) than typical AHL lactonases (ca. > 1 mM). X-ray crystal structures of AidC alone, and with the product N-hexanoyl-L-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multi-angle light scattering. The structures reveal two atypical features as compared to previously characterized AHL lactonases: a ‘kinked’ α-helix that forms part of a closed binding pocket which provides affinity and enforces selectivity for AHL substrates, and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. Implications for the catalytic mechanism of AHL lactonases are discussed.