title: Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro.
abstract: 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 (kcat/KM) 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 kcat/KMvalues 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 kcat/KMvalues >107M-1min-1with 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.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Moshe
authors.initials: M
authors.lastname: Goldsmith
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Eckstein
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Yacov
authors.initials: Y
authors.lastname: Ashani
authors.affiliation: Institute for Protein Design and Department of Biochemistry, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Per
authors.initials: P
authors.lastname: Greisen
authors.affiliation: Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Haim
authors.initials: H
authors.lastname: Leader
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Joel L
authors.initials: JL
authors.lastname: Sussman
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Nidhi
authors.initials: N
authors.lastname: Aggarwal
authors.affiliation: Institute for Protein Design and Department of Biochemistry, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Sergey
authors.initials: S
authors.lastname: Ovchinnikov
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Dan S
authors.initials: DS
authors.lastname: Tawfik
authors.affiliation: Institute for Protein Design and Department of Biochemistry, University of Washington, Seattle, WA, 98195, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: Thiermann
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany. franzworek@bundeswehr.org.
authors.forename: Franz
authors.initials: F
authors.lastname: Worek
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002999:Clone Cells; D019295:Computational Biology; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D057166:High-Throughput Screening Assays; D008658:Inactivation, Metabolic; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D009154:Mutation; D000067397:Nerve Agents / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism*; D019151:Peptide Library; D010575:Pesticides / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D044345:Phosphoric Triester Hydrolases / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D011549:Pseudomonas / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D000067397:Nerve Agents; D009943:Organophosphorus Compounds; D019151:Peptide Library; D010575:Pesticides; D011993:Recombinant Fusion Proteins; D044345:Phosphoric Triester Hydrolases
keywords: Bioscavenger; Detoxification; Mutant; Organophosphate hydrolase; Organophosphorus compounds; Phosphotriesterase