Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro

• DOI: 10.1007/s00204-015-1626-2
• Publication Date: 2015-11-26T07:07:13Z
• Authors: Moshe Goldsmith; Simone Eckstein; Yacov Ashani; Per Greisen; Haim Leader; Joel L. Sussman; Nidhi Aggarwal; Sergey Ovchinnikov; Dan S. Tawfik; David Baker; Horst Thiermann; Franz Worek
• Keywords: 0301 basic medicine; Molecular Structure; Recombinant Fusion Proteins; Computational Biology; Protein Engineering; Clone Cells; High-Throughput Screening Assays; 3. Good health; Molecular Docking Simulation; 03 medical and health sciences; Organophosphorus Compounds; Phosphoric Triester Hydrolases; Amino Acid Substitution; Bacterial Proteins; Peptide Library; Pseudomonas; Inactivation, Metabolic; Mutation; Biocatalysis; Escherichia coli; Directed Molecular Evolution; Pesticides; Nerve Agents

The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M-1 min-1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.