Paraoxonase-1 (PON1) is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP) compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we “humanized” an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence. 1. Introduction Paraoxonase-1 (PON1) is a mammalian serum protein, the activity of which is related to cardiovascular health and the toxicology of organophosphorus (OP) compounds [1–3]. PON1 is thought to be synthesized mostly in the liver, and it is associated with high-density lipoproteins (HDLs) in serum [4]. The exact function of PON1 is not known, but it is an efficient hydrolase of lactones and esters and an inefficient hydrolase of OP compounds, including pesticide metabolites such as paraoxon (from parathion) and chlorpyrifos oxon, and nerve agents such as sarin, tabun, and VX [1, 5]. Increased PON1 activity appears to be related to lower levels of oxidation of low-density lipoprotein (LDL) particles, and its hydrolytic activity has been suggested to be directed at oxidized fatty acids and homocysteine thiolactone [6–8]. Its increased activity has been shown to be related to decreased atherosclerosis, and it has been implicated in mechanisms of cholesterol efflux [9, 10]. PON1 also efficiently hydrolyzes bacterial lactones involved in quorum sensing, and it may contribute to innate immunity through this activity [11]. Although the hydrolysis of OP compounds is almost certainly a promiscuous activity of the enzyme, it contributes to the susceptibility to OP intoxication [12], and PON1 has been suggested as a lead
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