Catalytic and Conformational Stability Improvement of Organophosphorus Hydrolase

有机磷水解酶的催化和构象稳定性改进

• 批准号: 9904635
• 负责人: James Wild
• 金额: $ 37.97万
• 依托单位: Texas A&M AgriLife Research
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9904635&HistoricalAwards=false
• 关键词: Catalytic; Conformational; Stability; Improvement; Organophosphorus

9904635 Wild The widespread use of organophosphorus (OP) neurotoxins poses an environmental challenge for remediation. The organophosphate degrading gene of Pseudomonas diminuta encodes the metalloenzyme organophosphorus hydrolase (OPH) which catalyzes the breakdown of OP neurotoxins including the phosphorus-fluorine bond of Soman and Sarin, and the phosphothioates such as VX, although at very low rates. The ability to improve the rate of P-S bond hydrolysis will have implications in bioremediation of extremely toxic neurotoxins. The broad substrate specificity and hydrolytic efficiency (108-109 s-1M-1 for paraoxon) make OPH suitable for bioremediation technologies. The rate of phosphothioate bond hydrolysis has been improved up to thirty-fold using site-directed mutagenesis. However, these enzymes show significant reductions in conformational stability. This research has three goals: 1) Further enhancement of enzyme activity and specificity. 2) Determination of the three-dimensional structure of these enzymes. 3) Investigation of the thermodynamic stability of catalytically important variants and modification of enzyme properties for enhanced stability. Site-specific mutagenesis, protein crystallography and equilibrium and thermal denaturation studies are the basic techniques used. The requirement for new remediation technologies and the remarkable characteristics of OPH combine to form a biochemical framework for the design of new and improved OPH enzymes.Federal, state, and local authorities are concerned about the use of nerve agents in acts of domestic terrorism and are desperately searching for useful countermeasures. A remarkable enzyme isolated from a common soil bacterium, Pseudomonas diminuta, can detoxify some of the most deadly chemical warfare agents, including VX and Sarin, the nerve agent used in the Tokyo subway incident. However, the enzyme is not very efficient in degrading these compounds. This research is tailored to significantly enhance the catalytic capability of OPH for organophosphorus neurotoxins suitable for bioremediation technologies.

有机磷（OP）神经毒素的广泛使用对补救造成了环境挑战。 缺陷假单胞菌的有机磷降解基因编码金属酶有机磷水解酶（OPH），其催化OP神经毒素（包括梭曼和沙林的磷-氟键）和硫代磷酸酯（如VX）的分解，尽管速率非常低。 提高P-S键水解速率的能力将对剧毒神经毒素的生物修复产生影响。 广泛的底物特异性和水解效率（对氧磷为108-109 s-1 M-1）使OPH适合于生物修复技术。使用定点诱变，硫代磷酸酯键水解的速率已经提高了30倍。 然而，这些酶显示构象稳定性显著降低。 本研究有三个目的：1）进一步提高酶的活性和特异性。 2)确定这些酶的三维结构。 3)研究催化重要变体的热力学稳定性，并改进酶的性质以提高稳定性。定点诱变、蛋白质晶体学和平衡及热变性研究是所使用的基本技术。 对新的修复技术的要求和OPH的显著特性结合联合收割机，形成了设计新的和改进的OPH酶的生化框架。联邦、州和地方当局都很关注在国内恐怖主义行为中使用神经毒剂，并在拼命寻找有用的对策。 从一种常见的土壤细菌中分离出的一种非凡的酶，缺陷假单胞菌，可以解毒一些最致命的化学战剂，包括VX和沙林，东京地铁事件中使用的神经毒剂。 然而，该酶在降解这些化合物方面不是非常有效。 这项研究是量身定制的，以显着提高催化能力的OPH有机磷神经毒素适合生物修复技术。