The Evolutionary Origin and Potential of Newly Recruited Enzymes

描述（由申请人提供）：酶的进化是地球上生命出现的关键因素，并且其随后的分化占据了广泛的生态位。大多数酶都具有令人印象深刻的催化能力，这些能力已经经过了38亿年的进化。我们对新酶进化的早期阶段感兴趣。这项申请涉及的酶似乎是最近才被招募来在Sphingobium chlorophenolicum（一种从受到五氯苯酚严重污染的土壤中分离出来的细菌）的五氯苯酚降解途径中发挥新的功能的。S.看来，chlorophenolicum利用至少两种先前存在的途径中的酶，拼凑出了一条功能不佳的降解五氯苯酚的途径。这个实验系统为我们提供了一个窗口，在一个新的代谢途径的演变在一个非常早期的阶段，在这个过程中。本申请侧重于该途径中限制五氯苯酚降解途径通量的最初三种酶。五氯苯酚羟化酶催化五氯苯酚的羟化反应，生成毒性中间体四氯苯醌（TCBQ）。TCBQ还原酶催化TCBQ还原为四氯氢醌（TCHQ）。TCHQ脱卤酶催化两个连续的还原脱卤反应，除去氯，使环被切割。我们的目标是1）获得PCP羟化酶和TCHQ脱卤酶的结构; 2）探索TCBQ还原酶的进化起源和可能的额外功能; 3）进化所有三种酶的改进版本;以及4）确定改进的酶如何影响细菌降解PCP的能力。这项工作的结果将是更好地了解新酶可能产生的来源，以及在新酶进化的初始阶段实现的催化质量。公共卫生相关性：我们将更好地了解降解有毒污染物的途径的演变前景，以及通过招募解毒抗生素的酶而产生抗生素耐药性的相关问题。此外，这项工作将为工程酶和细菌生产化学品和药物或在环境友好条件下降解污染物的努力提供信息。

DESCRIPTION (provided by applicant): The evolution of enzymes was a critical element of the emergence of life on Earth and its subsequent divergence to occupy a vast range of ecological niches. Most enzymes have impressive catalytic capabilities that have been honed over 3.8 billion years of evolution. We are interested in the early stages of evolution of new enzymes. This application addresses enzymes that appear to have been recently recruited to serve new functions in the pathway for degradation of pentachlorophenol (PCP) in Sphingobium chlorophenolicum, a bacterium isolated from soil heavily contaminated with PCP. S. chlorophenolicum appears to have patched together a poorly functioning pathway for degradation of PCP using enzymes from at least two previously existing pathways. This experimental system provides us with a window on the evolution of a new metabolic pathway at a very early stage in the process. This application focuses on the initial three enzymes in the pathway that limit the flux through the PCP degradation pathway. PCP hydroxylase catalyzes the hydroxylation of PCP to form the toxic intermediate, tetrachlorobenzoquinone (TCBQ). TCBQ reductase catalyzes the reduction of TCBQ to tetrachlorohydroquinone (TCHQ). TCHQ dehalogenase catalyzes two successive reductive dehalogenation reactions that remove chlorines, allowing the ring to be cleaved. Our aims are 1) to obtain structures of PCP hydroxylase and TCHQ dehalogenase; 2) to explore the evolutionary origin and a possible additional function of TCBQ reductase; 3) to evolve improved versions of all three enzymes; and 4) to determine how the improved enzymes affect the ability of the bacterium to degrade PCP. The outcome of this work will be a better understanding of the sources from which new enzymes can arise, and the quality of catalysis achieved during the initial stages of evolution of a new enzyme. PUBLIC HEALTH RELEVANCE: We will better understand the prospects for evolution of pathways to degrade toxic pollutants and the problems associated with development of antibiotic resistance by recruitment of enzymes that detoxify antibiotics. In addition, this work will inform efforts to engineer enzymes and bacteria to produce chemicals and pharmaceuticals or to degrade pollutants under environmentally benign conditions.