Dramatic Differences in Organophosphorus Hydrolase Activity between Human and Chimeric Recombinant Mammalian Paraoxonase-1 Enzymes

Physiology and Immunology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, Maryland 21010-5400, USA.
Biochemistry (Impact Factor: 3.02). 09/2009; 48(43):10416-22. DOI: 10.1021/bi901161b
Source: PubMed


Human serum paraoxonase-1 (HuPON1) has the capacity to hydrolyze aryl esters, lactones, oxidized phospholipids, and organophosphorus (OP) compounds. HuPON1 and bacterially expressed chimeric recombinant PON1s (G2E6 and G3C9) differ by multiple amino acids, none of which are in the putative enzyme active site. To address the importance of these amino acid differences, the abilities of HuPON1, G2E6, G3C9, and several variants to hydrolyze phenyl acetate, paraoxon, and V-type OP nerve agents were examined. HuPON1 and G2E6 have a 10-fold greater catalytic efficiency toward phenyl acetate than G3C9. In contrast, bacterial PON1s are better able to promote hydrolysis of paraoxon, whereas HuPON1 is considerably better at catalyzing the hydrolysis of nerve agents VX and VR. These studies demonstrate that mutations distant from the active site of PON1 have large and unpredictable effects on the substrate specificities and possibly the hydrolytic mechanisms of HuPON1, G2E6, and G3C9. The replacement of residue H115 in the putative active site with tryptophan (H115W) has highly disparate effects on HuPON1 and G2E6. In HuPON1, variant H115W loses the ability to hydrolyze VR but has improved activity toward paraoxon and VX. The H115W variant of G2E6 has paraoxonase activity similar to that of wild-type G2E6, modest activity with phenyl acetate and VR, and enhanced VX hydrolysis. VR inhibits H115W HuPON1 competitively when paraoxon is the substrate and noncompetitively when VX is the substrate. We have identified the first variant of HuPON1, H115W, that displays significantly enhanced catalytic activity against an authentic V-type nerve agent.

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    • "The crystal structure for a recombinant PON1 (rPON1) has been elucidated, and has been shown to be a six-bladed ␤-propeller, which in the central tunnel contains two calcium ions, one of which is essential for enzyme activity and the other for stability (Harel et al., 2004). However, this rPON1 differs from the human enzyme by at least 51 aminoacids (Trovaslet-Leroy et al., 2011), and the OP esterase activity of human PON1 and rPON1 are substantially different (Otto et al., 2009). Thus, the 3D structure of human PON1 remains to be determined. "
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    ABSTRACT: Paraoxonase (PON1) is an A-esterase capable of hydrolyzing the active metabolites (oxons) of a number of organophosphorus (OP) insecticides such as parathion, diazinon and chlorpyrifos. PON1 activity is highest in liver and in plasma. Human PON1 displays two polymorphisms in the coding region (Q192R and L55M) and several polymorphisms in the promoter and the 3'-UTR regions. The Q192R polymorphism imparts differential catalytic activity toward some OP substrates, while the polymorphism at position -108 (C/T) is the major contributor of differences in the levels of PON1 expression. Both contribute to determining an individual's PON1 "status". Animal studies have shown that PON1 is an important determinant of OP toxicity. Administration of exogenous PON1 to rats or mice protects them from the toxicity of specific OPs. PON1 knockout mice display a high sensitivity to the toxicity of diazoxon and chlorpyrifos oxon, but not of paraoxon. In vitro catalytic efficiencies of purified PON(192) alloforms for hydrolysis of specific oxon substrates accurately predict the degree of in vivo protection afforded by each isoform. Evidence is slowly emerging that a low PON1 status may increase susceptibility to OP toxicity in humans. Low PON1 activity may also contribute to the developmental toxicity and neurotoxicity of OPs, as shown by animal and human studies.
    Toxicology 07/2012; 307. DOI:10.1016/j.tox.2012.07.011 · 3.62 Impact Factor
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    • "We chose to humanize the recently reported 4E9 variant (Figure 1) [15], which has very high activity against the cyclosarin (GF) analog CMP, using the same strategy used to generate g2e6p-huPON1. Because the substrate specificities of huPON1 and G2E6 PON1 are quite different despite essentially identical active sites, it was not clear if humanization could yield an active enzyme [5]. Here we show that humanization of 4E9 was successful and suggests a path forward for improved therapeutics based on engineered PON1 variants. "
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    ABSTRACT: 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.
    06/2012; 2012(2):610937. DOI:10.1155/2012/610937
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    • "Stock solutions were prepared at 0.9 mg mL−1 in 5 mM Tris, 10 mM CaCl2 at pH 7.4 and stored at −70°C. A modified Ellman-based colorimetric assay [16] was used to assess hydrolysis activity. Catalytic efficiency (kcat/KM) was calculated using the Michaelis-Menten equation with the approximation that the substrate concentration was much less than KM. "
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    ABSTRACT: Human Serum paraoxonase 1 (HuPON1) is an enzyme that has been shown to hydrolyze a variety of chemicals including the nerve agent VX. While wildtype HuPON1 does not exhibit sufficient activity against VX to be used as an in vivo countermeasure, it has been suggested that increasing HuPON1's organophosphorous hydrolase activity by one or two orders of magnitude would make the enzyme suitable for this purpose. The binding interaction between HuPON1 and VX has recently been modeled, but the mechanism for VX hydrolysis is still unknown. In this study, we created a transition state model for VX hydrolysis (VX(ts)) in water using quantum mechanical/molecular mechanical simulations, and docked the transition state model to 22 experimentally characterized HuPON1 variants using AutoDock Vina. The HuPON1-VX(ts) complexes were grouped by reaction mechanism using a novel clustering procedure. The average Vina interaction energies for different clusters were compared to the experimentally determined activities of HuPON1 variants to determine which computational procedures best predict how well HuPON1 variants will hydrolyze VX. The analysis showed that only conformations which have the attacking hydroxyl group of VX(ts) coordinated by the sidechain oxygen of D269 have a significant correlation with experimental results. The results from this study can be used for further characterization of how HuPON1 hydrolyzes VX and design of HuPON1 variants with increased activity against VX.
    PLoS ONE 05/2011; 6(5):e20335. DOI:10.1371/journal.pone.0020335 · 3.23 Impact Factor
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