Catalytic and Conformational Stability Improvement of Organophosphorus Hydrolase

有机磷（OP）神经毒素的广泛使用对环境修复提出了挑战。小假单胞菌的有机磷酸盐降解基因编码金属酶有机磷水解酶（OPH），该酶催化OP神经毒素的分解，包括索曼和沙林的磷-氟键，以及磷硫酸盐，如VX，尽管速率很低。提高P-S键水解速率的能力将对极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（对对氧磷为108-109 s-1M-1）使OPH适合生物修复技术。使用位点定向诱变，磷硫酸键水解的速率提高了30倍。然而，这些酶的构象稳定性明显降低。本研究有三个目标：1)进一步提高酶的活性和特异性。2)测定这些酶的三维结构。3)研究催化重要变异体的热力学稳定性，并对酶的性质进行修饰以增强稳定性。位点特异性诱变、蛋白质晶体学、平衡和热变性研究是使用的基本技术。对新修复技术的要求和OPH的显著特性结合起来，形成了设计新的和改进的OPH酶的生化框架。联邦、州和地方当局都对在国内恐怖主义活动中使用神经毒剂感到担忧，并正在拼命寻找有效的对策。从一种常见的土壤细菌小假单胞菌（Pseudomonas diminuta）中分离出一种非凡的酶，可以解毒一些最致命的化学战剂，包括VX和东京地铁事件中使用的神经毒剂沙林（Sarin）。然而，这种酶在降解这些化合物方面不是很有效。本研究旨在显著提高OPH对适合生物修复技术的有机磷神经毒素的催化能力。

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.