Organohalide pollutant detoxification using engineered reductive dehalogenases

还原脱卤酶（rdaase）是有机卤化物呼吸的关键酶，是由一种独特的细菌进行的。rda酶依赖于钴胺，能够通过还原选择性地切割碳-卤素键。因此，含有RDase的生物已被作为一种潜在的生物修复工具，用于清理由于处置不当而积累的有机卤化物污染的厌氧场所。rda酶可分为两类：i)典型呼吸脱卤酶，其在有机卤化物呼吸过程中使用卤化化合物作为最终电子受体；ii)分解代谢呼吸脱卤酶，其发生在非有机卤化物呼吸细菌的分解代谢途径中。分解代谢rda酶往往是耐氧的，这表明它们是未来机制研究和生物修复应用的更理想的靶点。在底物/配体复杂晶体结构的基础上，提出了一系列机制，包括远程机制和短程机制/内球机制。因此，目前尚不清楚什么是适当的底物定位和B12减少的基本驱动因素。利用一系列分解代谢还原脱卤酶，我们将确定支配底物特异性的是什么，并探索合理重新利用它的范围。我们将建立b12介导的底物还原机制（使用晶体方法）和NAD(P)H介导的b12还原机制（使用光谱方法），以提供对催化的完整机制理解。我们将利用在曼彻斯特获得的基本知识来指导多伦多环境相关有机卤化物的体内生物修复。该研究的更基本方面将在曼彻斯特的MIB进行，利用当地在酶学、蛋白质工程和生物技术方面的优势。体内生物修复研究将在多伦多BioZone进行，利用当地化学工程方面的专业知识。我们建议学生将花12个月的安置在第三年爱德华兹实验室在BioZone。该项目既适合世界一流的生物科学领域，也适合工业生物技术领域。

The reductive dehalogenases (RDases) are key enzymes in organohalide respiration, which is performed by a unique subset of bacteria. The RDases are cobalamin-dependent and able to selectively cleave carbon-halogen bonds through reduction. As a result, RDase containing organisms have been targeted as a potential bioremediation tool for the clean-up of contaminated anaerobic sites where organohalides have accumulated, often due to improper disposal. The RDases can be split into two classes: i) the canonical respiratory dehalogenases, which use a halogenated compound as a final electron acceptor during organohalide respiration and ii) the catabolic respiratory dehalogenases that occur in the catabolic pathways of non-organohalide respiring bacteria [3]. The catabolic RDase enzymes tend to be oxygen-tolerant, suggesting these are more desirable targets for future mechanistic studies and bioremediation applications. A range of mechanisms have been proposed including long range versus short range/inner sphere mechanisms on the basis of substrate/ligand complex crystal structures. It thus remains unclear what are the fundamental drivers of appropriate substrate positioning and B12 reduction. Making use of a range of catabolic reductive dehalogenases we will determine what governs substrate specificity and explore the scope to rationally repurpose this. We will establish both the mechanism of B12-mediated substrate reduction (using in crystallo approaches) and NAD(P)H mediated B12-reduction (using spectroscopic approaches) to provide a complete mechanistic understanding of catalysis. We will use fundamental understanding gained at Manchester to guide in vivo bioremediation of environmentally relevant organohalides at Toronto. The more fundamental aspects of the study will be carried out at Manchester in the MIB taking advantage of the local strength in enzymology, protein engineering and biotechnology. The in vivo bioremediation studies will be carried out in Toronta at BioZone taking advantage of the local expertise in chemical engineering. We propose the student will spend a ~12 month placement in the 3rd year at the Edwards lab in BioZone. The project fits both within the world class bioscience as well as industrial biotechnology areas.