Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 34134, Republic of Korea.
Agency for Defense Development, P.O. Box 35, Yuseong, Daejeon 34186, Republic of Korea.
J Microbiol Biotechnol. 2021 Jan 28;31(1):144-153. doi: 10.4014/jmb.2006.06044.
Organophosphorus nerve agents (OPNAs), including both G- and V-type nerve agents such as sarin, soman, tabun and VX, are extremely neurotoxic organophosphorus compounds. Catalytic bioscavengers capable of hydrolyzing OPNAs are under development because of the low protective effects and adverse side effects of chemical antidotes to OPNA poisoning. However, these bioscavengers have certain limitations for practical application, including low catalytic activity and narrow specificity. In this study, we generated a fusion-hybrid form of engineered recombinant human paraoxonase 1 (rePON1) and bacterial organophosphorus hydrolase (OPH), referred to as GV-hybrids, using a flexible linker to develop more promising catalytic bioscavengers against a broad range of OPNAs. These GV-hybrids were able to synergistically hydrolyze both G-type OPNA analogs (paraoxon: 1.7 ~ 193.7-fold, -nitrophenyl diphenyl phosphate (PNPDPP): 2.3 ~ 33.0-fold and diisopropyl fluorophosphates (DFP): 1.4 ~ 22.8-fold) and V-type OPNA analogs (demeton-Smethyl (DSM): 1.9 ~ 34.6-fold and malathion: 1.1 ~ 4.2-fold above) better than their individual enzyme forms. Among the GV-hybrid clones, the GV7 clone showed remarkable improvements in the catalytic activity toward both G-type OPNA analogs (/ (10 M min): 59.8 ± 0.06 (paraoxon), 5.2 ± 0.02 (PNPDPP) and 47.0 ± 6.0 (DFP)) and V-type OPNA analogs (/ (M min): 504.3 ± 48.5 (DSM) and 1324.0 ± 47.5 (malathion)). In conclusion, we developed GV-hybrid forms of rePON1 and bacterial OPH mutants as effective and suitable catalytic bioscavengers to hydrolyze a broad range of OPNA analogs.
有机磷神经毒剂（OPNAs），包括 G 型和 V 型神经毒剂如沙林、梭曼、塔崩和 VX，是极其神经毒性的有机磷化合物。由于对 OPNA 中毒的化学解毒剂的保护作用低和不良反应，能够水解 OPNAs 的催化生物清除剂正在开发中。然而，这些生物清除剂在实际应用中具有一定的局限性，包括催化活性低和特异性窄。在这项研究中，我们使用柔性接头生成了工程重组人对氧磷酶 1（rePON1）和细菌有机磷水解酶（OPH）的融合杂种形式，称为 GV-杂种，以开发针对广泛 OPNA 的更有前途的催化生物清除剂。这些 GV-杂种能够协同水解 G 型 OPNA 类似物（对氧磷：1.7193.7 倍，-硝基苯基二苯基磷酸酯（PNPDPP）：2.333.0 倍和二异丙基氟磷酸酯（DFP）：1.422.8 倍）和 V 型 OPNA 类似物（地美松-S-甲基（DSM）：1.934.6 倍和马拉硫磷：1.1~4.2 倍），优于它们的单一酶形式。在 GV-杂种克隆中，GV7 克隆在对 G 型 OPNA 类似物（/（10Mmin）：59.8±0.06（对氧磷）、5.2±0.02（PNPDPP）和 47.0±6.0（DFP））和 V 型 OPNA 类似物（/（Mmin）：504.3±48.5（DSM）和 1324.0±47.5（马拉硫磷））的催化活性方面表现出显著的改善。总之，我们开发了 rePON1 和细菌 OPH 突变体的 GV-杂种形式，作为有效和合适的催化生物清除剂，用于水解广泛的 OPNA 类似物。
