MAA Isomerase to Tetrachlorohydroquinone Dehalogenase: Evolution in Action

0077569 Copley释放到环境中的异生物质化合物对微生物施加选择性压力，促进新的分解代谢途径的进化。 招募预先存在的酶以提供新的功能是微生物组装新的分解代谢途径的一个重要策略。 鞘氨醇单胞菌chlorophenolica似乎已经招募马来酰乙酰乙酸（MAA）异构酶作为四氯氢醌（TCHQ）脱卤酶在有毒的异源生物农药，五氯苯酚的生物降解过程中。 MAA异构酶在酪氨酸催化过程中催化谷胱甘肽依赖的双键异构化。 TCHQ脱卤酶用氢原子取代TCHQ和三氯对苯二酚上的氯取代基，这一过程导致两个谷胱甘肽分子氧化成谷胱甘肽二硫化物。 虽然这些反应是显着不同的，TCHQ脱卤酶具有相当大的序列与已知的MAA异构酶的活性位点区域的同一性，并具有显着的异构酶活性。 最初的努力将针对一个严格的分析的祖蛋白的功能，包括阐明的反应机制和活性位点残基的作用的识别。 随后，将对开发和优化改变的活性所需的突变进行详细分析，重点是了解每个改变的残基的功能。最后，在体外进化技术将被用来尝试通过减轻限制TCHQ脱卤酶的催化能力的底物抑制来进化更有效的酶。 这项工作将提供新的见解，以应对环境毒素的新酶的进化。它还将产生关于蛋白质支架的通用催化能力适应显著不同目的的重要信息，这些信息应该为有兴趣开发新催化剂的蛋白质工程师提供灵感。 最后，它将提供一种改进的TCHQ脱卤酶的基因，该基因可用于改造改良的S.氯酚类化合物，其降解五氯苯酚的能力得到增强。

0077569CopleyXenobiotic compounds released to the environment exert selective pressures on microorganisms that promote the evolution of new catabolic pathways. Recruitment of pre-existing enzymes to serve new functions is one important strategy by which microorganisms assemble new catabolic pathways. Sphingomonas chlorophenolica appears to have recruited maleylacetoacetate (MAA) isomerase to serve as a tetrachlorohydroquinone (TCHQ) dehalogenase during biodegradation of the toxic xenobiotic pesticide, pentachlorophenol. MAA isomerase catalyzes the glutathione-dependent isomerization of a double bond during the catabolism of tyrosine. TCHQ dehalogenase replaces chlorine substituents on TCHQ and trichlorohydroquinone with hydrogen atoms, a process which results in the oxidation of two molecules of glutathione to glutathione disulfide. Although these reactions are remarkably different, TCHQ dehalogenase has considerable sequence identity with known MAA isomerases in the active site region and has substantial isomerase activity. Initial efforts will be directed at a rigorous analysis of the function of the progenitor protein, including elucidation of the reaction mechanism and identification of the roles of active site residues. Subsequently, a detailed analysis of the mutations required to develop and optimize the altered activity will be carried out, with emphasis on understanding the function of each residue that is changed. Finally, in vitro evolution techniques will be used to try to evolve a more effective enzyme by alleviating the substrate inhibition that limits the catalytic ability of TCHQ dehalogenase. This work will provide new insights into the evolution of new enzymes in response to environmental toxins. It will also yield important information about the adaptation of the generic catalytic capabilities of a protein scaffold for dramatically different purposes that should provide inspiration for protein engineers interested in developing new catalysts. Finally, it will provide a gene for an improved TCHQ dehalogenase that should be useful in the engineering of an improved strain of S. chlorophenolica with an enhanced ability to degrade pentachlorophenol.