Catalytic and Conformational Stability Improvement of Organophosphorus Hydrolase

9904635野生有机磷(OP)神经毒素的广泛使用对补救工作构成了环境挑战。微小假单胞菌的有机磷降解基因编码金属有机磷水解酶(OPH)，该酶催化分解OP神经毒素，包括Soman和Sarin的磷-氟键，以及VX等硫代磷酸盐，尽管速率很低。提高P-S键水解率的能力将对剧毒神经毒素的生物修复具有重要意义。广泛的底物专一性和水解率(对氧磷的水解率为10~8~10~9 S~(-1)·m~(-1))使OPH适合于生物修复技术。通过定点突变，硫代磷键的水解率提高了30倍。然而，这些酶的构象稳定性显著降低。本研究有三个目标：1)进一步提高酶的活性和特异性。2)这些酶的三维结构的测定。3)研究重要催化变异体的热力学稳定性和为提高稳定性而对酶的性质进行修改。定点突变、蛋白质结晶学、平衡和热变性研究是使用的基本技术。对新的补救技术的要求和OPH的显著特征结合在一起，形成了设计新的和改进的OPH酶的生化框架。联邦、州和地方当局对在国内恐怖主义行为中使用神经毒剂感到担忧，并正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引人注目的酶，可以为一些最致命的化学战剂解毒，包括VX和东京地铁事故中使用的神经毒剂Sarin。然而，这种酶在降解这些化合物方面并不是很有效。这项研究旨在显著提高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.