Benign Synthesis, Bioremediation and Safer Chemical Design: Factors in P450-Substrate Interactions

9710129琼斯这项建议详细说明了在追求绿色化学的过程中使用细胞色素P450酶的研究。对P450-配体相互作用性质的研究将被用作生物催化剂，以设计一种具有工业重要性的羧酸-2-乙基己酸乙酯的创新合成。生物催化途径将导致污染的预防，因为它将消除在合成过程中对有毒和致癌重金属的需求。这一合成被选为P450酶家族如何被用作一般生物催化剂的模型。这些研究将用于开发可用于设计更安全的化学品的预测方法。预测方法将使人们能够对新化学品开发中的生物激活潜力做出快速决策。因此，毒性较小的化学品可以设计成新用途的化学品，并快速识别潜在危险的化学品。细胞色素P450是一个普遍存在的酶家族，它催化分子氧还原为活性很强的单氧物种。这种酶系统负责内源和外源化学物质的代谢。细胞色素P450(CYP)酶家族被称为已知用途最广的生物催化剂。这一特性是一把双刃剑，使CYP酶家族成为多种化学物质的解毒剂，以及负责产生有毒代谢物的主要酶系统。因此，虽然酶在生物合成反应中很重要，但在良性化学物质对致癌物和毒素的生物激活中也很重要。这项建议的总体目标是了解控制细胞色素P450与配体相互作用的结构因素。将构建P450cam的活性位点突变体，并对其进行运动学鉴定。来自这些突变体的结构信息，从X射线结晶学和分子动力学研究中获得，将确定底物与P450cam结合的重要因素。这些信息可用于设计P450突变体以进行良性合成，设计更安全的化学品，以及设计用于生物修复的P450酶。虽然用于生物修复的细菌的开发不在这笔赠款的范围内，但在该项目期间获得的结果将直接适用于这一努力。此外，还将获得关于配体-酶相互作用机制的一般知识，这些知识可以应用于P450以外的酶系统。本研究的具体目标如下：(1)选育P450 cam活性位点突变体F87W、Y96W、L244A和T185F。(1.2)化学和动力学表征野生型和突变型酶氧化为工业重要产物2-乙基己酸的区域和立体专一性。(1.3)用分子动力学方法研究活性部位的酶-配体相互作用。(1.4)通过检查酶和酶-配体复合体的晶体结构来检验动力学模型。***

9710129 Jones This proposal details studies on the use of cytochrome P450 enzymes in the pursuit of green chemistry. Studies on the nature of P450-ligand interactions will be exploited as a biocatalyst to design an innovative synthesis for the industrially important carboxylic acid, 2-ethylhexanoate. The biocatalysis pathway will lead to pollution prevention since it will obviate the need for toxic and carcinogenic heavy metals in this synthesis. This synthesis was chosen as a model for how the P450 enzyme family can be exploited as a biocatalyst in general. These same studies will be used to develop predictive methodology that can be exploited in the design of safer chemicals. The predictive methodology will allow for rapid decision making about the potential of bioactivation in the development of new chemicals. Thus, less toxic chemicals can be designed into chemicals for new applications and potentially hazardous chemical identified rapidly. The cytochromes P450 are a ubiquitous family of enzymes that catalyze the reduction of molecular oxygen to a very reactive monooxygen species. This enzyme system is responsible for the metabolism of both endogenous and exogenous chemicals. The cytochrome P450 (CYP) enzyme family has been called the most versatile biological catalyst known. This trait is a double edged sword that makes the CYP enzyme family a diverse detoxifier of chemicals, as well as, the major enzyme system responsible for the production of toxic metabolites. Thus, while the enzymeis important in biosynthetic reactions, it is also important in bioactivation of benign chemicals to carcinogens and toxins. The general objective of this proposal is to understand the structural factors that govern cytochrome P450-ligand interactions. Active site mutants of P450cam will be constructed and kinetically characterized. Structural information from these mutants, gained from Xray crystallography and molecular dynamics studies, will define factors important in substrate binding to P450cam. This information can be used in the design of P450 mutants to perform benign synthesis, in the design of safer chemicals and in the design of P450 enzymes for bioremediation. While the development of bacteria for bioremediation is not within the scope of this grant, the results obtained during this project will be directly applicable to this endeavor. Furthermore, general knowledge about the mechanism of ligand- enzyme interactions, that can be applied to enzyme systems other than P450, will be obtained. The following are the specific objectives of this proposal: (1.1) Produce P450cam active-site mutants F87W, Y96W, L244A, and T185F. (1.2) Chemically and kinetically characterize regio- and stereospecificity of oxidations to the industrially important product 2-ethylhexanoic acid by the wild-type and mutant enzymes. (1.3) Study enzyme-ligand interactions at the active site using molecular dynamics. (1.4) Test the dynamics models by examination of crystal structures of the enzyme and enzyme-ligand complexes. ***