Probing the mechanism of enzyme-catalyzed reductive dehalogenation for bioremediation

探讨酶催化还原脱卤生物修复的机制

• 批准号: RGPIN-2022-04446
• 负责人: Edwards, Elizabeth
• 金额: $ 6.56万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=747254
• 关键词: Probing; mechanism; enzyme; catalyzed; reductive

Organohalide-respiring bacteria (OHRB) are a remarkable group of strictly anaerobic bacteria that use organic compounds with one or several halogen substituents as terminal electron acceptors for energy and growth. A wide variety of naturally occurring and man-made halogenated organic compounds are found in the environment, explaining the widespread distribution of OHRB. In fact, the discovery of this microbial metabolism over 20 years ago revolutionized bioremediation practice. Bioaugmentation, where an aquifer is inoculated with mixed microbial populations of specialized OHRB, is now commonly used for groundwater clean-up, particularly for chlorinated ethenes, ethanes and methanes -all common industrial solvents and widespread toxic pollutants. OHRB are broadly distributed within the Proteobacteria, Chloroflexi, and Firmicutes. Members of the Chloroflexi, particularly Dehalococcoides and Dehalogenimonas, and specific Firmicutes (Dehalobacter) are of exceptional interest because these genera are obligate organohalide respirers. They are ideal for bioremediation because their only metabolism is dehalogenation. The enzymes catalyzing the cleavage of a carbon-halogen bond in OHRB are known as reductive dehalogenases (RDases). RDases contains a vitamin B12 co-factor and two iron sulfur clusters. Thousands of putative sequences have been identified in (meta)genomes from environmental and culture samples, but only a few dozen have been partially characterized. Heterologous expression of functional reductive dehalogenases has been largely unsuccessful, hampering characterization. My research group has developed genomic and proteomic techniques and anaerobic enzyme assays adapted to fastidious anaerobic cultures used for bioremediation. We have managed to identify substrates for a few reductive dehalogenases in their native hosts in enrichment cultures. We developed a classification system to organize putative dehalogenase sequences into hundreds of different ortholog clusters. But the most exciting recent development has been the discovery that by co-expressing a vitamin B12 import pathway with a reductive dehalogenase we could produce active dechorinating enzymes in E. coli. This result finally opens the door to much easier characterization of this extraordinarily useful enzyme family, because we no longer need to produce the enzymes in their slow-growing and genetically intractable native host. Building from this recent finding, the focus of this Discovery Grant is to further mine this novel approach for heterologous expression to 1) rapidly assay the many uncharacterized enzymes on panels of organohalides to identify their substrates and inhibitors, 2) probe mechanism and substrate interactions through structural and mutational analyses, 3) engineer these enzymes for new substrates, including perhaps the problematic perfluorinated compounds, and 4) use this information to inform new remediation approaches.

有机卤素呼吸细菌（OHRB）是一类显著的严格厌氧细菌，它们利用含有一个或几个卤素取代基的有机化合物作为终端电子受体来获取能量和生长。在环境中发现了各种各样的天然和人造卤化有机化合物，这解释了OHRB的广泛分布。事实上，20多年前这种微生物代谢的发现彻底改变了生物修复实践。生物强化，即在含水层接种特殊OHRB的混合微生物种群，现在通常用于地下水清理，特别是氯代乙烯、乙烷和甲烷- -所有常见的工业溶剂和广泛存在的有毒污染物。OHRB广泛分布于变形菌门、绿菌门和厚壁菌门。氯氟菌门的成员，特别是去卤球菌门和去卤单胞菌门，以及特定的厚壁菌门（去卤杆菌门）特别令人感兴趣，因为这些属是专性有机卤化物呼吸者。它们是理想的生物修复，因为它们唯一的代谢是脱卤。催化OHRB中碳-卤素键断裂的酶被称为还原脱卤酶（rdaase）。rases含有维生素B12辅助因子和两个铁硫簇。从环境和培养样本的（元）基因组中已经确定了数千个假定的序列，但只有几十个已被部分表征。功能还原脱卤酶的异源表达在很大程度上是不成功的，阻碍了表征。我的研究小组已经开发了基因组和蛋白质组学技术和厌氧酶测定，适用于用于生物修复的苛刻厌氧培养。我们已经成功地在富集培养的原生宿主中鉴定了一些还原脱卤酶的底物。我们开发了一个分类系统，将假定的脱卤酶序列组织成数百个不同的同源群。但最近最令人兴奋的发现是，通过与还原性脱卤素酶共同表达维生素B12进口途径，我们可以在大肠杆菌中产生活性脱卤素酶。这一结果最终为更容易地描述这种非常有用的酶家族打开了大门，因为我们不再需要在它们生长缓慢且遗传上难以处理的原生宿主中生产酶。基于这一最新发现，本发现基金的重点是进一步挖掘这种异源表达的新方法：1)快速测定有机卤化物板上的许多未表征的酶，以确定其底物和抑制剂；2)通过结构和突变分析探测底物相互作用的机制；3)设计这些酶用于新的底物，包括可能存在问题的全氟化合物；4)利用这些信息为新的补救方法提供信息。