Probing the mechanism of enzyme-catalyzed reductive dehalogenation for bioremediation

有机卤化物呼吸细菌（Organohalide-respiring bacteria，OHRB）是一类以有机化合物为末端电子受体的厌氧细菌。在环境中发现了各种各样的天然和人造卤代有机化合物，解释了OHRB的广泛分布。事实上，这种微生物代谢的发现在20多年前彻底改变了生物修复实践。生物强化，即在含水层中接种专门的OHRB混合微生物种群，现在通常用于地下水净化，特别是用于氯代乙烯、乙烷和甲烷-所有这些都是常见的工业溶剂和广泛的有毒污染物。 OHRB广泛分布于变形菌门、绿弯菌门和厚壁菌门。Chloroflexi的成员，特别是Dehalococcoides和Dehalimonas，以及特定的Firmicutes（Dehalimonas）特别令人感兴趣，因为这些属是专性有机卤化物呼吸者。它们是生物修复的理想选择，因为它们唯一的代谢是脱卤。催化OHRB中碳-卤键裂解的酶被称为还原脱卤酶（RDases）。RDases含有维生素B12辅因子和两个铁硫簇。在环境和文化样本的（Meta）基因组中已经确定了数千个推定的序列，但只有几十个被部分表征。功能性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法，适用于用于生物修复的苛刻厌氧培养物。我们已经成功地确定了一些还原脱卤酶在其本地主机在富集文化的基板。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直系同源物簇。但最近最令人兴奋的进展是发现通过共表达维生素B12输入途径和还原性脱卤酶，我们可以在大肠杆菌中产生活性脱卤酶。杆菌这一结果最终为更容易地表征这种非常有用的酶家族打开了大门，因为我们不再需要在生长缓慢且遗传上难以控制的天然宿主中生产酶。 基于这一最新发现，该发现资助的重点是进一步挖掘这种用于异源表达的新方法，以1）快速测定有机卤化物面板上的许多未表征的酶，以鉴定其底物和抑制剂，2）通过结构和突变分析的探针机制和底物相互作用，3）针对新底物设计这些酶，可能包括有问题的全氟化合物，以及4）利用这些信息为新的补救方法提供信息。

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.