pubs.acs.org/Biochemistry Published on Web 06/04/2009 r 2009 American Chemical Society 6644 Biochemistry 2009, 48, 6644–6654 DOI: 10.1021/bi900583y Directed Evolution of Serum Paraoxonase PON3 by Family Shuffling and Ancestor/Consensus Mutagenesis, and Its Biochemical Characterization † Olga Khersonsky, ‡ Mira Rosenblat, § Lilly Toker, ) Shiri Yacobson, ‡ Adrian Hugenmatter, ‡ Israel Silman, ) Joel L. Sussman, ^ Michael Aviram, § and Dan S. Tawfik* ,‡ ‡ Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel, § Lipid Research Laboratory, Technion Faculty of Medicine, and Rambam Medical Center, Rappaport Family Institute for Research in the Medical Sciences, Haifa 31096, Israel, ) Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel, and ^ Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel Received April 6, 2009 ABSTRACT: Serum paraoxonases (PONs) are calcium-dependent lactonases with anti-atherogenic and detoxification functions. Here we describe the directed evolution and characterization of recombinant variants of serum paraoxonase PON3 that express in an active and soluble manner in Escherichia coli. These variants were obtained by combining family shuffling and phylogeny-based mutagenesis: the limited diversity of accessible, cloned PON3 genes was complemented by spiking the shuffling reaction with ancestor/ consensus mutations, mutations to residues that comprise the consensus or appear in the predicted ancestors of the PON family. We screened the resulting libraries for PON3’s lactonase activity while ensuring that the selected variants retained the substrate specificity of wild-type mammalian PON3s. The availability of highly stable, recombinant PON3 that is free of all other serum components enabled us to explore unknown biochemical features of PON3, including its binding to HDL particles, the effect of HDL on PON3’s stability and enzymatic activity, and ex vivo tests of its anti-atherogenic properties. Overall, it appears that PON3 possesses properties very similar to those of PON1: the enzyme’s lactonase activity is selectively stimulated by binding to apoAI-HDL, with a concomitant increase in its stability. PON3 also exhibits potentially anti- atherogenic functions, although at levels lower than those of PON1. Serum paraoxonases (PONs) 1 are calcium-dependent mam- malian enzymes that have been recently characterized as lipo- philic lactonases (1, 2). PONs exhibit a range of biologically important activities, such as organophosphate hydrolysis and protection against atherosclerosis, although their physiological substrate(s) remains unknown. PON1 is the only well-character- ized member of the family, the other members being PON2 and PON3. While PON1 is present mostly in the serum bound to HDL, PON2 present in all the tissues but not on HDL, while PON3 resides both on HDL and in various tissues (1, 3). PON3 has been studied much less than PON1. Its structure is unknown, nor was it engineered, primarily due to lack of ample sources of stable, bacterially expressed PON3 variants similar to those that permitted extensive exploration of PON1 (1, 4). It shares 65% amino acid identity with PON1 and exhibits sub- stantial lactonase activity. However, its promiscuous esterase and phosphotriesterase activities are much weaker than those of PON1 (1). Although it is known that PON3 has an anti- atherogenic potential (5-8), little is known about its biochemical properties. Before it became apparent that PONs are lactonases (1, 2), both PON1 and PON3 were subjected to directed evolution for Escherichia coli expression (4), using family shuffling and screens for the aryl-esterase and paraoxonase activities (that are now known to be promiscuous). In both cases, variants with a higher level of bacterial expression were selected. While in PON1 their enzymatic parameters fortuitously remained as they are for the wild type, serum-purified PON1 (including for lipophilic lactones that were not screened for at the time), improvement in bacterial expression of PON3 was accompanied by significant changes in substrate specificity, including >20-fold increases in aryl-esterase activity and >200-fold increases in phosphotriesterase activity. These recombinant PON3 variants were, therefore, unsuitable for mechanistic and biochemical studies aimed at improving our understanding of PON3’s native functions. We therefore aimed to perform directed evolution of PON3 while screening for the lactonase activity, assuming that such a procedure would yield the desired bacterial expression without changing the enzymatic properties. The lipophilic lactones † This work was supported by the EU network BioModularH2, the National Institutes of Health (Grant 81XWH-07-2-0020), and the Wolgin Prize to D.S.T. O.K. is supported by the Adams Fellowship Program of the Israel Academy of Sciences and Humanities. *To whom correspondence should be addressed: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel. Phone: þ972 8 934 3637. Fax: þ972 8 934 4118. E-mail: tawfik@weizmann.ac.il. 1 Abbreviations: PON, serum paraoxonase; HDL, high-density lipopro- tein; LDL, low-density lipoprotein; TBBL, 5-thiobutylbutyrolactone; TEBL, 5-thioethylbutyrolactone; THBL, 5-thiohexylbutyrolactone; WT, wild-type. Downloaded by JEWISH NATL & UNIV LIB ISRAEL on July 19, 2009 Published on June 4, 2009 on http://pubs.acs.org | doi: 10.1021/bi900583y