State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Joint International Research Laboratory of Metabolic & Developmental Sciences, Shanghai Jiao Tong University, Shanghai, China.
Appl Environ Microbiol. 2020 Dec 17;87(1). doi: 10.1128/AEM.01965-20.
Hexachlorobenzene (HCB), as one of the persistent organic pollutants (POPs) and a possible human carcinogen, is especially resistant to biodegradation. In this study, HcbA1A3, a distinct flavin-N5-peroxide-utilizing enzyme and the sole known naturally occurring aerobic HCB dechlorinase, was biochemically characterized. Its apparent preference for HCB in binding affinity revealed that HcbA1 could oxidize only HCB rather than less-chlorinated benzenes such as pentachlorobenzene and tetrachlorobenzenes. In addition, the crystal structure of HcbA1 and its complex with flavin mononucleotide (FMN) were resolved, revealing HcbA1 to be a new member of the bacterial luciferase-like family. A much smaller substrate-binding pocket of HcbA1 than is seen with its close homologues suggests a requirement of limited space for catalysis. In the active center, Tyr362 and Asp315 are necessary in maintaining the normal conformation of HcbA1, while Arg311, Arg314, Phe10, Val59, and Met12 are pivotal for the substrate affinity. They are supposed to place HCB at a productive orientation through multiple interactions. His17, with its close contact with the site of oxidation of HCB, probably fixes the target chlorine atom and stabilizes reaction intermediates. The enzymatic characteristics and crystal structures reported here provide new insights into the substrate specificity and catalytic mechanism of HcbA1, which paves the way for its rational engineering and application in the bioremediation of HCB-polluted environments. As an endocrine disrupter and possible carcinogen to human beings, hexachlorobenzene (HCB) is especially resistant to biodegradation, largely due to difficulty in its dechlorination. The lack of knowledge of HCB dechlorinases limits their application in bioremediation. Recently, an HCB monooxygenase, HcbA1A3, representing the only naturally occurring aerobic HCB dechlorinase known so far, was reported. Here, we report its biochemical and structural characterization, providing new insights into its substrate selectivity and catalytic mechanism. This research also increases our understanding of HCB dechlorinases and flavin-N5-peroxide-utilizing enzymes.
六氯苯（HCB）作为持久性有机污染物（POPs）之一和一种可能的人类致癌物，特别难以生物降解。在这项研究中，我们对一种独特的黄素-N5-过氧化物利用酶和唯一已知的天然好氧 HCB 脱氯酶 HcbA1A3 进行了生化特性分析。其在结合亲和力方面对 HCB 的明显偏好表明，HcbA1 只能氧化 HCB，而不能氧化五氯苯和四氯苯等较少氯化的苯。此外，还解析了 HcbA1 及其与黄素单核苷酸（FMN）复合物的晶体结构，表明 HcbA1 是细菌荧光素样家族的新成员。与近亲相比，HcbA1 的底物结合口袋要小得多，这表明催化需要有限的空间。在活性中心，Tyr362 和 Asp315 对于维持 HcbA1 的正常构象是必需的，而 Arg311、Arg314、Phe10、Val59 和 Met12 对于底物亲和力是至关重要的。它们通过多种相互作用将 HCB 置于有利的取向。His17 与 HCB 氧化部位紧密接触，可能固定靶氯原子并稳定反应中间体。这里报道的酶学特性和晶体结构为 HcbA1 的底物特异性和催化机制提供了新的见解，为其在 HCB 污染环境的生物修复中的合理工程化和应用铺平了道路。作为一种对人类具有内分泌干扰作用和可能致癌作用的物质，六氯苯（HCB）特别难以生物降解，这主要是由于其脱氯困难。缺乏 HCB 脱氯酶的知识限制了它们在生物修复中的应用。最近，报道了一种 HCB 单加氧酶 HcbA1A3，它代表了迄今为止已知的唯一天然好氧 HCB 脱氯酶。在这里，我们报告了它的生化和结构特性，为其底物选择性和催化机制提供了新的见解。这项研究也增加了我们对 HCB 脱氯酶和黄素-N5-过氧化物利用酶的理解。
