Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, TX 77842, USA.
Biochemistry. 2012 Aug 14;51(32):6463-75. doi: 10.1021/bi300811t. Epub 2012 Jul 31.
Phosphotriesterase (PTE) from soil bacteria is known for its ability to catalyze the detoxification of organophosphate pesticides and chemical warfare agents. Most of the organophosphate chemical warfare agents are a mixture of two stereoisomers at the phosphorus center, and the S(P)-enantiomers are significantly more toxic than the R(P)-enantiomers. In previous investigations, PTE variants were created through the manipulation of the substrate binding pockets and these mutants were shown to have greater catalytic activities for the detoxification of the more toxic S(P)-enantiomers of nerve agent analogues for GB, GD, GF, VX, and VR than the less toxic R(P)-enantiomers. In this investigation, alternate strategies were employed to discover additional PTE variants with significant improvements in catalytic activities relative to that of the wild-type enzyme. Screening and selection techniques were utilized to isolate PTE variants from randomized libraries and site specific modifications. The catalytic activities of these newly identified PTE variants toward the S(P)-enantiomers of chromophoric analogues of GB, GD, GF, VX, and VR have been improved up to 15000-fold relative to that of the wild-type enzyme. The X-ray crystal structures of the best PTE variants were determined. Characterization of these mutants with the authentic G-type nerve agents has confirmed the expected improvements in catalytic activity against the most toxic enantiomers of GB, GD, and GF. The values of k(cat)/K(m) for the H257Y/L303T (YT) mutant for the hydrolysis of GB, GD, and GF were determined to be 2 × 10(6), 5 × 10(5), and 8 × 10(5) M(-1) s(-1), respectively. The YT mutant is the most proficient enzyme reported thus far for the detoxification of G-type nerve agents. These results support a combinatorial strategy of rational design and directed evolution as a powerful tool for the discovery of more efficient enzymes for the detoxification of organophosphate nerve agents.
土壤细菌中的磷酸三酯酶 (PTE) 以催化解毒有机磷农药和化学战剂的能力而闻名。大多数有机磷化学战剂是磷中心两个对映异构体的混合物，S(P)-对映异构体的毒性比 R(P)-对映异构体大得多。在以前的研究中，通过操纵底物结合口袋来创建 PTE 变体，这些突变体被证明对神经毒剂类似物 GB、GD、GF、VX 和 VR 的更有毒 S(P)-对映异构体的解毒具有更高的催化活性，而对毒性较小的 R(P)-对映异构体则不然。在这项研究中，采用了替代策略来发现具有相对于野生型酶显著提高催化活性的其他 PTE 变体。筛选和选择技术用于从随机文库中分离 PTE 变体和定点修饰。这些新鉴定的 PTE 变体对 GB、GD、GF、VX 和 VR 的生色类似物的 S(P)-对映异构体的催化活性提高了 15000 倍以上。最佳 PTE 变体的 X 射线晶体结构已被确定。用真实的 G 型神经毒剂对这些突变体进行的表征证实了对 GB、GD 和 GF 的最有毒对映异构体的催化活性的预期提高。H257Y/L303T (YT) 突变体对 GB、GD 和 GF 水解的 k(cat)/K(m) 值分别确定为 2×10(6)、5×10(5)和 8×10(5)M(-1)s(-1)。YT 突变体是迄今为止报道的用于解毒 G 型神经毒剂的最有效酶。这些结果支持了合理设计和定向进化的组合策略，这是发现用于解毒有机磷神经毒剂的更有效酶的有力工具。
