title: Altering the substrate specificity of methyl parathion hydrolase with directed evolution.
abstract: Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important. Metalloenzymes such as methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 hold great promise as bioremediators as they are able to hydrolyze a wide range of OPs. MPH is highly efficient towards methyl parathion (1 × 10(6) s(-1) M(-1)), but its activity towards other OPs is more modest. Thus, site saturation mutagenesis (SSM) and DNA shuffling were performed to find mutants with improved activities on ethyl paraxon (6.1 × 10(3) s(-1) M(-1)). SSM was performed on nine residues lining the active site. Several mutants with modest activity enhancement towards ethyl paraoxon were isolated and used as templates for DNA shuffling. Ultimately, 14 multiple-site mutants with enhanced activity were isolated. One mutant, R2F3, exhibited a nearly 100-fold increase in the kcat/Km value for ethyl paraoxon (5.9 × 10(5) s(-1) M(-1)). These studies highlight the 'plasticity' of the MPH active site that facilitates the fine-tuning of its active site towards specific substrates with only minor changes required. MPH is thus an ideal candidate for the development of an enzyme-based bioremediation system.
authors: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
authors: Tee-Kheang
authors: TK
authors: Ng
authors: School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.
authors: Lawrence R
authors: LR
authors: Gahan
authors: School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.
authors: Gerhard
authors: G
authors: Schenk
authors: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia. Electronic address: ollis@rsc.anu.edu.au.
authors: David L
authors: DL
authors: Ollis
mesh_terms: D004390:Chlorpyrifos / Q000031:analogs & derivatives / Q000737:chemistry; D032621:DNA Shuffling; D019020:Directed Molecular Evolution; D006868:Hydrolysis; D007700:Kinetics; D008743:Methyl Parathion / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D010755:Organophosphates / Q000737:chemistry*; D010261:Paraoxon / Q000031:analogs & derivatives / Q000737:chemistry; D010278:Parathion / Q000737:chemistry; D010575:Pesticides / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D010755:Organophosphates; D010575:Pesticides; C121104:ethylparaoxon; D008743:Methyl Parathion; D010278:Parathion; C495424:methyl parathion hydrolase, Pseudomonas; D010744:Phosphoric Monoester Hydrolases; D004390:Chlorpyrifos; D010261:Paraoxon; C008480:methylparaoxon
keywords: Bioremediation; Directed evolution; Methyl parathion hydrolase; Organophosphate pesticides; Substrate specificity