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

9710129 Jones该提案详细研究了在追求绿色化学中使用细胞色素P450酶的情况。对p450 -配体相互作用性质的研究将被用作生物催化剂，用于设计工业上重要的羧酸- 2-乙基己酸酯的创新合成。生物催化途径将导致污染预防，因为它将避免在合成中需要有毒和致癌的重金属。该合成被选为P450酶家族如何被利用作为生物催化剂的模型。这些同样的研究将用于开发可用于设计更安全化学品的预测方法。预测方法将允许对新化学品开发中生物活化的潜力进行快速决策。因此，毒性较低的化学品可以被设计成用于新应用的化学品，潜在的危险化学品可以被迅速识别。细胞色素P450是一个普遍存在的酶家族，催化分子氧还原为非常活跃的单氧物种。这种酶系统负责内源性和外源性化学物质的代谢。细胞色素P450 （CYP）酶家族被称为已知最通用的生物催化剂。这一特性是一把双刃剑，它使CYP酶家族成为多种化学物质的解毒剂，同时也是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，但它在良性化学物质对致癌物和毒素的生物活化中也很重要。本提案的总体目标是了解控制细胞色素p450 -配体相互作用的结构因素。构建P450cam活性位点突变体并进行动力学表征。从x射线晶体学和分子动力学研究中获得的这些突变体的结构信息将确定底物与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此资助范围内，但在此项目中获得的结果将直接适用于此努力。此外，将获得有关配体-酶相互作用机制的一般知识，可应用于P450以外的酶系统。以下是本提案的具体目标：（1.1）产生P450cam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工业重要产品2-乙基己酸的区域和立体特异性。（1.3）利用分子动力学方法研究酶与配体在活性位点的相互作用。（1.4）通过检测酶和酶配体复合物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. ***