Probing the mechanism of enzyme-catalyzed reductive dehalogenation for bioremediation

点击翻译按钮获取中文摘要

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.