title: Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state.
abstract: Phosphotriesterase from Pseudomonas diminuta (PTE) is an extremely efficient metalloenzyme that hydrolyses a variety of compounds including organophosphorus nerve agents. Study of PTE has been hampered by difficulties with efficient expression of the recombinant form of this highly interesting and potentially useful enzyme. We identified a low-level esterolytic activity of PTE and then screened PTE gene libraries for improvements in 2-naphthyl acetate hydrolysis. However, the attempt to evolve this promiscuous esterase activity led to a variant (S5) containing three point mutations that resulted in a 20-fold increase in functional expression. Interestingly, the zinc holoenzyme form of S5 appears to be more sensitive than wild-type PTE to both thermal denaturation and addition of metal chelators. Higher functional expression of the S5 variant seems to lie in a higher stability of the metal-free apoenzyme. The results obtained in this work point out another-and often overlooked-possible determinant of protein expression and purification yields, i.e. the stability of intermediates during protein folding and processing.
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: C
authors.initials: C
authors.lastname: Roodveldt
authors.forename: D S
authors.initials: DS
authors.lastname: Tawfik
mesh_terms: D002614:Chelating Agents / Q000737:chemistry; D019020:Directed Molecular Evolution; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011489:Protein Denaturation; D011549:Pseudomonas / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D002614:Chelating Agents; D011993:Recombinant Fusion Proteins; D044345:Phosphoric Triester Hydrolases