Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization

Amir Aharoni, Leonid Gaidukov, Shai Yagur, Lilly Toker, Israel Silman, Dan S. Tawfik

Research output: Contribution to journalArticlepeer-review

251 Scopus citations

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.

Original languageEnglish
Pages (from-to)482-487
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number2
DOIs
StatePublished - 13 Jan 2004
Externally publishedYes

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