title: Engineering the Organophosphorus Acid Anhydrolase Enzyme for Increased Catalytic Efficiency and Broadened Stereospecificity on Russian VX.
abstract: 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.
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Courtney M
authors.initials: CM
authors.lastname: Daczkowski
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia , Athens, Georgia 30602, United States.|U.S. Army Reserve 377th Sustainment Command Detachment 8 , Aberdeen Proving Ground, Maryland 21010-5424, United States.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Pegan
authors.affiliation: U.S. Army Edgewood Chemical Biological Center , Aberdeen Proving Ground, Maryland 21010-5424, United States.
authors.forename: Steven P
authors.initials: SP
authors.lastname: Harvey
mesh_terms: D020591:Alteromonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D043303:Aryldialkylphosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009946:Organothiophosphorus Compounds / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D009946:Organothiophosphorus Compounds; D043303:Aryldialkylphosphatase; C451779:S-(N,N-diethylaminoethyl) isobutyl methylphosphothiolate