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

良性合成、生物修复和更安全的化学设计：P450-底物相互作用的因素

• 批准号: 9710129
• 负责人: Jeffrey Jones
• 金额: $ 20.68万
• 依托单位: University of Rochester
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-10-01 至 1999-02-11
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9710129&HistoricalAwards=false
• 关键词: Benign; Synthesis; Bioremediation; Safer; Chemical

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. ***

9710129 Jones该提案详细介绍了在追求绿色化学中使用细胞色素P450酶的研究。 P450-配体相互作用的性质的研究将被利用作为生物催化剂，以设计一个创新的合成工业上重要的羧酸，2-乙基己酸。 生物催化途径将导致污染预防，因为它将消除在这种合成中对有毒和致癌重金属的需要。 选择这种合成作为P450酶家族如何通常可用作生物催化剂的模型。 这些相同的研究将用于开发预测方法，可用于设计更安全的化学品。 预测方法将允许在新化学品的开发中对生物活化的潜力做出快速决策。 因此，毒性较小的化学品可以设计成新应用的化学品，并迅速识别潜在的危险化学品。 细胞色素P450是一个普遍存在的酶家族，其催化分子氧还原成非常活泼的单氧物种。 该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，使酶家族成为多种化学物质的解毒剂，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，但它在将良性化学物质生物活化为致癌物和毒素方面也很重要。 这个建议的总体目标是了解的结构因素，管理细胞色素P450配体的相互作用。 将构建P450 cam的活性位点突变体并进行动力学表征。 从这些突变体的结构信息，从X射线晶体学和分子动力学研究中获得的，将定义重要的因素在底物结合P450 CAM。 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本资助范围内，但在本项目期间获得的结果将直接适用于这一奋进。 此外，将获得关于配体-酶相互作用机制的一般知识，其可应用于P450以外的酶系统。 以下是这项建议的具体目标： (1.1)产生P450 cam活性位点突变体F87 W、Y 96 W、L244 A和T185 F。 (1.2)从化学和动力学上表征野生型和突变型酶对工业重要产物2-乙基己酸氧化的区域和立体特异性。 (1.3)用分子动力学研究酶与配体在活性位点的相互作用。 （1.4） 通过对晶体的检测来检验动力学模型 酶和酶-配体复合物的结构。 ***