Ng Tee-Kheang, Gahan Lawrence R, Schenk Gerhard, Ollis David L
Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.
Arch Biochem Biophys. 2015 May 1;573:59-68. doi: 10.1016/j.abb.2015.03.012. Epub 2015 Mar 19.
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.
许多有机磷酸酯（OPs）被用作农业杀虫剂。由于它们对乙酰胆碱酯酶的抑制作用，会对健康构成严重危害。因此，对被OPs污染的水和土壤进行解毒很重要。来自假单胞菌属WBC - 3的金属酶，如甲基对硫磷水解酶（MPH），作为生物修复剂具有很大的潜力，因为它们能够水解多种OPs。MPH对甲基对硫磷具有高效性（1×10⁶ s⁻¹ M⁻¹），但其对其他OPs的活性则较为一般。因此，进行了位点饱和诱变（SSM）和DNA改组，以寻找对乙基对氧磷（6.1×10³ s⁻¹ M⁻¹）活性有所提高的突变体。对活性位点周围的9个残基进行了SSM。分离出了几个对乙基对氧磷活性略有增强的突变体，并将其用作DNA改组的模板。最终，分离出了14个活性增强的多位点突变体。一个突变体R2F3对乙基对氧磷的kcat/Km值增加了近100倍（5.9×10⁵ s⁻¹ M⁻¹）。这些研究突出了MPH活性位点的“可塑性”，即仅需进行微小改变就能促进其活性位点针对特定底物进行微调。因此，MPH是开发基于酶的生物修复系统的理想候选者。
