title: Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization.
abstract: Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate (OP) detoxification and in prevention of atherosclerosis. However, their structure and mechanism of action are poorly understood. PONs seem like jacks-of-all-trades, acting on a very wide range of substrates, most of which are of no physiological relevance. Family shuffling and screening lead to the first PON variants that express in a soluble and active form in Escherichia coli. We describe variants with kinetic parameters similar to those reported for PONs purified from sera and others that show dramatically increased activities. In particular, we have evolved PON1 variants with OP-hydrolyzing activities 40-fold higher than wild type and a specificity switch of >2,000-fold, producing PONs specialized for OP rather than ester hydrolysis. Analysis of the newly evolved variants provides insights into the evolutionary relationships between different family members.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Amir
authors.initials: A
authors.lastname: Aharoni
authors.forename: Leonid
authors.initials: L
authors.lastname: Gaidukov
authors.forename: Shai
authors.initials: S
authors.lastname: Yagur
authors.forename: Lilly
authors.initials: L
authors.lastname: Toker
authors.forename: Israel
authors.initials: I
authors.lastname: Silman
authors.forename: Dan S
authors.initials: DS
authors.lastname: Tawfik
mesh_terms: D043303:Aryldialkylphosphatase / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human