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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
Published on: August 21, 2018
Structural and mutational studies of organophosphorus hydrolase reveal a cryptic and functional allosteric-binding
Janet K Grimsley1, Barbara Calamini, James R Wild
1Biochemistry and Biophysics Department, Texas A&M University, College Station, TX 77843-2128, USA.
Abstract:
Organophosphorus hydrolase detoxifies a broad range of organophosphate pesticides and the chemical warfare agents (CWAs) sarin and VX. Previously, rational genetic engineering produced OPH variants with 30-fold enhancements in the hydrolysis of CWA and their analogs. One interesting variant (H254R) in which the histidine at position 254 was changed to an arginine showed a 4-fold increase in the hydrolysis of demetonS (VX analog), a 14-fold decrease with paraoxon (an insecticide), and a 183-fold decrease with DFP (sarin analog). The three-dimensional structure of this enzyme at 1.9A resolution with the inhibitor, diethyl 4-methylbenzylphosphonate (EBP), revealed that the inhibitor did not bind at the active site, but bound exclusively into a well-defined surface pocket 12 A away from the active site. This structural feature was accompanied by non-competitive inhibition of paraoxon hydrolysis by EBP with H254R, in contrast to the native enzyme, which showed competitive inhibition. These parallel structure-function characteristics identify a functional, allosteric site on the surface of this enzyme.
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