Altering the substrate specificity of methyl parathion hydrolase with directed evolution

Tee Kheang Ng, Lawrence R. Gahan, Gerhard Schenk, David L. Ollis*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    28 Citations (Scopus)

    Abstract

    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 × 106 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 × 103 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 × 105 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.

    Original languageEnglish
    Article number6949
    Pages (from-to)59-68
    Number of pages10
    JournalArchives of Biochemistry and Biophysics
    Volume573
    DOIs
    Publication statusPublished - 1 May 2015

    Fingerprint

    Dive into the research topics of 'Altering the substrate specificity of methyl parathion hydrolase with directed evolution'. Together they form a unique fingerprint.

    Cite this