Enzymatic Hydrolysis of Organophosphate Esters

有机磷酸酯的酶水解

• 批准号: 9235651
• 负责人: Frank M. Raushel
• 金额: $ 27.93万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9235651
• 关键词: Active Sites; Agriculture; Amino Acid Sequence; Bacteria; Binding; Biological; Chemical Warfare Agents; Chemicals; Complement; Crystallization; Detection; Drug Metabolic Detoxication; Enzyme Inhibitor Drugs; Enzymes; Esters; Event; Exhibits; Flame Retardants; Household; Hydrolysis; Insecticides; Isotopes; Kinetics; Label; Laboratories; Libraries; Ligands; Measurement; Methods; Monitor; Mutagenesis; Organophosphates; Oxygen; Pesticides; Phosphoric Triester Hydrolases; Phosphorus; Plasticizers; Procedures; Prodrugs; Property; Pseudomonas; Reaction; Research; Resolution; Series; Shapes; Site-Directed Mutagenesis; Soman; Structure; Toxic effect; Variant; Virus Diseases; X ray diffraction analysis; X-Ray Diffraction; catalyst; chemical reaction; combinatorial; design; enzyme mechanism; experimental study; inhibitor/antagonist; mutant; nerve agent; novel; screening; three dimensional structure

The primary objective for the research described in this proposal is the elucidation of the chemical reaction mechanism and three-dimensional structure of a novel phosphotriesterase recently identified from the bacterium Spingobium sp. strain TCM1 (Sb-PTE). The high toxicity of many organophosphate triesters has been exploited as the active component in many commercial agricultural and household insecticides and as ultra-potent chemical warfare agents. Other, less toxic, organophosphate compounds have been widely utilized as flame retardants, plasticizers, and as prodrugs for viral infections. The catalytic properties and three-dimensional structure of the wild- type Sb-PTE are significantly different from those exhibited by the phosphotriesterase from Pseudomonas diminuta (Pd-PTE) or any other enzyme identified to date. We propose to utilize this enzyme as a template for the design and creation of new biological catalysts that can be exploited for the detection, destruction, and detoxification of toxic organophosphate nerve agents that are currently being used as agricultural and household insecticides, plasticizers, and chemical warfare agents. The chemical mechanism for this enzyme will be elucidated by determining the stereochemical course of the reaction with chiral substrates and by monitoring the fate of 18-oxygen labels in the substrate and enzyme. These experiments will be complemented by measurement of heavy atom isotope effects, determination of the substrate/activity profile, and the identification of potent enzyme inhibitors. In preliminary experiments we have succeeded in the crystallization of Sb-PTE and determination of its three dimensional structure by X-ray diffraction methods. Further structures will be pursued in a focused attempt to determine the mode of substrate binding within the active site of this enzyme. These structures will be utilized as a guide for the design and creation of novel enzyme variants with unique substrate profiles. Rational and combinatorial libraries of mutant enzymes will be constructed and those variants with enhanced catalytic proficiency for the hydrolysis of toxic organophosphates will be identified through unique screening and selection procedures. The changes in the amino acid sequence of the Sb-PTE mutants will be correlated with enhancements in the catalytic properties and alterations in the structure within the active site.

本提案中所描述的研究的主要目的是阐明