bi5b00624_si_001.pdf (278.81 kB)

Engineering the Organophosphorus Acid Anhydrolase Enzyme for Increased Catalytic Efficiency and Broadened Stereospecificity on Russian VX

Download (278.81 kB)
journal contribution
posted on 20.10.2015, 00:00 by Courtney M. Daczkowski, Scott D. Pegan, Steven P. Harvey
The enzyme organophosphorus acid anhydrolase (OPAA), from Alteromonas sp. JD6.5, has been shown to rapidly catalyze the hydrolysis of a number of toxic organophosphorus compounds, including several G-type chemical nerve agents. The enzyme was cloned into Escherichia coli and can be produced up to approximately 50% of cellular protein. There have been no previous reports of OPAA activity on VR {Russian VX, O-isobutyl S-[2-(diethylamino)­ethyl] methylphosphonothioate}, and our studies reported here show that wild-type OPAA has poor catalytic efficacy toward VR. However, via application of a structurally aided protein engineering approach, significant improvements in catalytic efficiency were realized via optimization of the small pocket within the OPAA’s substrate-binding site. This optimization involved alterations at only three amino acid sites resulting in a 30-fold increase in catalytic efficiency toward racemic VR, with a strong stereospecificity toward the P­(+) enantiomer. X-ray structures of this mutant as well as one of its predecessors provide potential structural rationales for their effect on the OPAA active site. Additionally, a fourth mutation at a site near the small pocket was found to relax the stereospecificity of the OPAA enzyme. Thus, it allows the altered enzyme to effectively process both VR enantiomers and should be a useful genetic background in which to seek further improvements in OPAA VR activity.