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.

有机甲虫吸引的细菌（OHRB）是一组显着的厌氧菌细菌，它们使用有机化合物，其中一种或几种卤素取代基作为末端电子受体，用于能量和生长。在环境中发现了各种各样的自然存在和人造卤代有机化合物，解释了OHRB的广泛分布。实际上，20年前这种微生物代谢的发现彻底改变了生物修复实践。生物调节含水层与专门OHRB的混合微生物种群接种的生物层，现在通常用于地下水清理，尤其是用于氯化乙烯，乙烯和甲烷 - 所有常见的工业溶剂和广泛的有毒污染物。 OHRB广泛分布在蛋白质，叶绿体和企业中。氯氟诺夫的成员，尤其是去足球和脱甲虫虫以及特定的企业（脱甲杆菌）的成员具有非凡的兴趣，因为这些属是强大的Organohalide呼吸器。它们是生物修复的理想选择，因为它们唯一的代谢是去alogenation。在OHRB中催化碳释放键的裂解的酶称为还原性去催化酶（RDases）。 RDases含有维生素B12副因素和两个铁硫簇。在环境和培养样品的（元）基因组中已经确定了数千个推定的序列，但仅部分表征了几十个。功能还原性去催化酶的异源表达在很大程度上没有成功，阻碍了特征。 我的研究小组开发了基因组和蛋白质组学技术和厌氧酶测定法，适用于用于生物修复的挑剔的厌氧培养物。我们设法鉴定了其在富集培养物中的原生宿主中一些还原性脱核酶的底物。我们开发了一个分类系统，将推定的脱核酶序列组织成数百个不同的直系同源群。但是，最近最令人兴奋的发展是发现，通过将维生素B12进口途径与还原性脱核酶共同表达，我们可以在大肠杆菌中产生活跃的脱氢酶。最终，这个结果为这种非常有用的酶家族的表征更容易地打开了大门，因为我们不再需要在其缓慢生长且遗传性的本机宿主中产生这些酶。 从这一发现的重点开始，这项发现的重点是进一步挖掘这种新颖的表达方法1）迅速在有机甲降低的有机盐上迅速测定许多未表征的酶，以识别其底物和抑制剂，2）探针机制和底物相互作用，通过结构和突变分析，包括新的组合，包括新的问题，包括新的问题，包括新的问题，包括新的问题，包括新的问题。使用此信息为新的补救方法提供信息。