Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides.
Organophosphorus hydrolase (OPH, also known as phosphotriesterase) is a bacterial enzyme that is capable of degrading a wide range of neurotoxic organophosphate nerve agents. Directed evolution has been used to generate one variant (22A11) with up to 25-fold improved hydrolysis of methyl parathion. Surprisingly, this variant also degraded all other substrates (paraoxon, parathion and coumaphos) tested 2- to 10-fold faster. Since only one mutation (H257Y) is directly located in the active site, site-directed mutagenesis and saturation mutagenesis were used to identify the role of the other distal substitutions (A14T, A80V, K185R, H257Y, I274N) on substrate specificity and activity. Sequential site-directed mutagenesis indicated that K185R and I274N are the most important substitutions, leading to an improvement not only in the hydrolysis of methyl parathion but also the overall hydrolysis rate of all other substrates tested. Using structural modeling, these two mutations were shown to favor the formation of hydrogen bonds with nearby residues, resulting in structural changes that could alter the overall substrate hydrolysis.
Mee-Hie Cho
Catherine
C
Department of Chemical and Environmental Engineering, University of California, Riverside, 92521, USA.
Mulchandani
Ashok
A
Chen
Wilfred
W
D019943:Amino Acid Substitution; D043303:Aryldialkylphosphatase / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D014644:Genetic Variation; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009943:Organophosphorus Compounds; D010575:Pesticides / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D009943:Organophosphorus Compounds; D010575:Pesticides; D043303:Aryldialkylphosphatase