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.

Fig. 1.

Directed evolution of PON1 for soluble expression in E. coli. (A) Paraoxonase activity of PON1 variants in the crude E. coli lysate calculated per milligram of E. coli cells. Activity is shown for the wild-type trx-HuPON1 (nil), trx-RabPON1, trx-rePON1 variants G1A5 and G1C4 from the first round and G2D6 and G2E6 from the second round, and rePON1 variant G3C9 (devoid of all tags) from the third round of evolution. (B) SDS/PAGE (12%) of the crude cell lysates as numbered in A.

Fig. 2.

Improvements in catalytic specificity (k_cat/K_M) toward various substrates of variants of trx-rePON3 from the different rounds of evolution (with respect to wild-type trx-RabPON3). G1A7 and G1B11 are first-generation variants, G2C2 is a second-generation variant, and G3A5, G3G3, and G3H9 are third-generation variants. The kinetic parameters are presented in Tables 2 and 9.

Fig. 3.

Michaelis–Menten plots for the hydrolysis of DEPCyC (A) and phenylacetate (B) by rePON1 variant G3C9 and its variant G3C9.49 that displays greatly enhanced OP-hydrolyzing activity. Enzyme concentrations are 7.5 × 10^–8 M and 5.6 × 10^–9 Min A and 5.2 × 10^–9 M and 3.4 × 10^–7 Min B for G3C9 and G3C9.49, respectively. The kinetic parameters derived from the fits are presented in Table 3.