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.

本提案中描述的研究的主要目标是阐明 近期新型磷酸三酯酶的化学反应机理及三维结构 从 Spingobium sp. 细菌中鉴定出来。菌株 TCM1 (Sb-PTE)。许多药物毒性较大 有机磷酸三酯已被用作许多商业中的活性成分 农业和家用杀虫剂以及超强化学战剂。其他毒性较小的， 有机磷酸酯化合物被广泛用作阻燃剂、增塑剂等 用于病毒感染的前药。野生材料的催化性能和三维结构 Sb-PTE 型与磷酸三酯酶所表现出的显着不同 Pseudomonas diminuta (Pd-PTE) 或迄今为止已鉴定的任何其他酶。我们建议利用这个 酶作为设计和创造新生物催化剂的模板，可用于 检测、破坏和解毒目前已发现的有毒有机磷神经毒剂 用作农业和家用杀虫剂、增塑剂和化学战剂。这 通过确定该酶的立体化学过程，将阐明该酶的化学机制 与手性底物的反应并通过监测底物中 18-氧标记的命运， 酶。这些实验将通过重原子同位素效应的测量得到补充， 底物/活性谱的测定，以及有效酶抑制剂的鉴定。在 初步实验成功实现了Sb-PTE的结晶并测定了其含量 通过 X 射线衍射方法测量三维结构。进一步的结构将在 重点尝试确定该酶活性位点内底物结合的模式。 这些结构将用作设计和创建新型酶变体的指南 独特的基材轮廓。将构建突变酶的合理和组合文库 以及那些对有毒有机磷酸酯水解具有增强催化能力的变体 将通过独特的筛选和选择程序来确定。氨基酸的变化 Sb-PTE 突变体的序列将与催化性能的增强相关 活性位点内结构的改变。