A 5000-Fold Increase in the Specificity of a Bacterial Phosphotriesterase for Malathion through Combinatorial Active Site Mutagenesis

Abstract

Phosphotriesterases (PTEs) have been isolated from a range of bacterial species, including Agrobcaterium radiobacter (PTEAr), and are efficient enzymes with broad substrate ranges. The turnover rate of PTEAr for the common organophosphorous insecticide malathion is lower than expected based on its physical properties; principally the pka of its leaving group. In this study, we rationalise the turnover rate of PTEAr for malathion using computational docking of the substrate into a high resolution crystal structure of the enzyme, suggesting that malathion is too large for the PTEAr binding pocket. Protein engineering through combinatorial active site saturation testing (CASTing) was then used to increase the rate of malathion turnover. Variants from a CASTing library in which Ser308 and Tyr309 were mutated yielded variants with increased activity towards malathion. The most active PTEAr variant carried Ser308Leu and Tyr309Ala substitutions, which resulted in a ca. 5000-fold increase in k cat/K M for malathion. X-ray crystal structures for the PTEAr Ser308Leu\Tyr309Ala variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky Tyr309 residue with the smaller alanine residue.

Enzymatic Bioremediation of Organophosphate Compounds—Progress and Remaining Challenges

“…PTE shows high catalytic activity toward a number of pesticides but only moderate activity toward nerve agents such as sarin, cyclosarin, and VX. The environmental persistence of V-agents such as VX has led several research teams such as those of Frank Raushel of Texas A&M University and others to invest heavily in mutational studies of this enzyme to improve activity toward VX and other substrates (Briseno-Roa et al, 2011; Tsai et al, 2012; Bigley et al, 2013, 2015; Naqvi et al, 2014; Bigely et al, 2019). Recently, Khersonsky et al developed a robust automated method which led to generation of PTE with a remarkable >1,000-fold improvement in nerve-agent hydrolysis (Khersonsky et al, 2018).…”

Recent advances in enzyme promiscuity

“…The catalytic activity of PTE from A. radiobacter, for the common organophosphorous insecticide malathion was enhanced by changing serine to leucine at position 308 and tyrosine to alanine at position 309, which resulted in 5000-fold increase in Kcat/Km value. X-ray crystal structures for the above variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky tyrosine residue with the smaller alanine residue [45].…”

Synergistic rhizosphere degradation of γ-hexachlorocyclohexane (lindane) through the combinatorial plant-fungal action

“…Lindane usually degrades under aerobic and anaerobic environments, but it can be mineralized only in aerobic condition [ 14 , 15 ]. Many bacteria has been reported for lindane degradation [ 15 – 18 ] and many scientist had attributed this to their tolerance and development of enzyme and genetic mechanisms to mineralize the pesticide over the years [ 15 ].…”