Chimeric Recombinant Mammalian Paraoxonase-1 Enzymes

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. Organophosphorus (OP) nerve agents are among the most toxic chemical substances identified (1). These compounds exert toxicity by readily binding to acetylcholinesterase (AChE) at the active site serine and inhibiting the ability of AChE to terminate cholinergic nerve transmissions (2). Existing pharmacologic treatments available to counteract the immediate effects of OP nerve agent intoxication, such as atropine, oximes, and diazepam, enhance survival but do not prevent performance deficits, behavioral incapacitation, loss of consciousness, or possible permanent brain damage (3). Current research has focused on the development of human butyrylcholinesterase as a stoichiometric bioscavenger to remove OP compounds from circulation before they can reach their physiological target (4-6). In an effort to identify a human protein that can catalyze the hydrolysis of OP nerve agents, we have focused our attention on human serum paraoxonase-1 (HuPON1) (7). HuPON1 is an HDL-associated enzyme that can catalyze the hydrolysis of a diverse group of substrates, including aryl esters, lactones, oxidized phospholipids, and OP compounds (8-12). Although the catalytic activity of HuPON1 toward OP nerve agents is too low to afford significant in vivo protection over butyrylcholinesterase, protein engineering could be used to increase the rate of nerve agent hydrolysis by the enzyme. On the basis of theoretical calculations, the catalytic efficiency ofHuPON1must be equal to or greater than 10M s and theKM must be less than 10 μM for the enzyme to act in vivo as a highly efficient catalytic bioscavenger of OP nerve agents (13). Functional HuPON1 has been notoriously difficult to express and purify in large quantities, despite attempts made in a variety of expression systems. For example, functional HuPON1 has been successfully produced from Escherichia coli, but in low yields (14) (T. J. Magliery et al., unpublished data). Using directed evolution via gene shuffling of human, rabbit, mouse, and rat PON1 genes, Aharoni et al. (15) expressed functional chimeric recombinant PON1 enzymes (G2E6 and G3C9, among others) in high yield in bacteria, thereby creating an opportunity to utilize high-throughput screening approaches to isolate variants with altered enzymatic function. Successful bacterial expression of the G2E6 enzyme enabled large-scale production of functional PON1 that was used in crystallization studies. As predicted by homology with DFPase (12), the X-ray structure of the G2E6 crystal revealed a 6-fold β-propeller protein centrally arranged around two calcium ions (16, 17). On the basis of this structure, Tawfik and colleagues proposed theH115-H134 dyad within the putative active site as the catalytic machinery of the enzyme (16, 18, 19). Other studies suggest that this mechanism This work was supported byNIHCounterACTCenter of Excellence Grant U54 NS058183 [to D.E.L. (Center PI), D.M.C., and T.J.M.] and by the Defense Threat Reduction Agency;Joint Science and Technology Office, Medical S&T Division (to D.E.L.). *To whom correspondence should be addressed. Telephone: (410) 436-3525. Fax: (410) 436-8377. E-mail: david.lenz@us.army.mil. Abbreviations: HuPON1, human serum paraoxonase-1; OP, organophosphorus; AChE, acetylcholinesterase; DTNB, 5,50-dithiobis(2-nitrobenzoic acid); PDB, Protein Data Bank. D ow nl oa de d by U S A R M Y M E D IC A L R E S M A T A M R M C o n O ct ob er 3 0, 2 00 9 | h ttp :// pu bs .a cs .o rg P ub lic at io n D at e (W eb ): S ep te m be r 18 , 2 00 9 | d oi : 1 0. 10 21 /b i9 01 16 1b Article Biochemistry, Vol. 48, No. 43, 2009 10417 may be correct only for describing lactone hydrolysis, which may be the native substrate of PON1 (12, 17-20). Because no crystal structures for HuPON1 or other variants have been reported, the structure of G2E6 has been used to approximate these enzymes (21). However, the amino acid composition of PON1 clone G2E6 differs from that of native HuPON1 in 59 amino acids, and fromG3C9 in 20 amino acids; in each case, the substitutions are distributed throughout the total of 355 residues (Figure 1 and Figure S1 of the Supporting Information). It is important to consider what effects the amino acid differences between these enzymesmayhave on hydrolysis of the same substrates. The goal of this study was to examine the specificities of HuPON1, G2E6, G3C9, and several variants with respect to the hydrolysis of phenyl acetate and paraoxon, as well as of nerve agents VX (ethyl {2-[di(propan-2-yl)amino]ethylsulfanyl}methylphosphinate) and VR {N,N-diethyl-2-[methyl(2-methylpropoxy)phosphoryl]sulfanylethanamine (seeFigure 2)}. In this paper, we show that human and bacterially expressed forms of PON1 have very different enzymatic activities with respect to phenyl acetate, paraoxon, and V-type nerve agents. While G2E6 and G3C9 exhibit improved hydrolysis of paraoxon compared to that of HuPON1, HuPON1 exhibits significantly better turnover of nerve agents VX and VR. Of the five variants examined, only the H115W variant of either HuPON1 or G2E6 enhanced catalytic efficiency for the hydrolysis of paraoxon and VX. EXPERIMENTAL PROCEDURES Expression of HuPON1. cDNAs encoding wild-type and H115W, S193A, S193G, R214Q, and S193A/R214Q variant HuPON1 proteins [with the Q192 allele and a C-terminal six histidine (6-His) tag] were cloned into pcDNA3.1 and transiently transfected into human embryonic kidney 293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Media were harvested two days post-transfection, filtered, and stored at 4 C. Expression of HuPON1 and variants was confirmed by Western analysis using a mouse monoclonal anti-HuPON1 antibody kindly provided by R. James (University Hospital of Geneva, Geneva, Switzerland). HuPON1 protein concentrations were determined from quantitative Western blot analyses using densitometry (Un-Scan-It version 5.1, Silk Scientific Corp., Orem, UT) with a HuPON1 standard at a known concentration (Randox Life Sciences, Antrim, U.K.). Expression and Purification of Bacterially Expressed PON1s. G3C9, G2E6, and the H115W, S193A, and R214Q variants of G2E6 were expressed and purified essentially as described previously (15, 16). G3C9 was expressed with a Cterminal 6-His tag, whereas G2E6 and variants were expressed as thioredoxin fusion proteins with N-terminal 6-His tags. The enzymes were expressed in Origami B (DE3) cells (Novagen, Madison, WI). When bacterial cultures reached an A600 of 0.8, they were induced with 0.1 mM IPTG for 3 h. Harvested cells were resuspended in lysis buffer [50 mMTris-HCl, 50 mMNaCl, 1 mM CaCl2, and 0.1 mM dithiothreitol (pH 8.0)] and extruded through a syringe needle. After sonication, the lysate was incubated with 0.1% Tergitol NP-10 (Sigma-Aldrich, St. Louis, MO) with shaking at 4 C for 150 min. Ni-NTA resin (Qiagen, Valencia, CA) was added to the lysate and themixture shaken for 4 h. The resin was washed with activity buffer [50 mM Tris-HCl (pH 8), 50 mM NaCl, 1 mM CaCl2, and 0.1% Tergitol NP-10], including 10 mM imidazole followed by a wash with activity buffer supplemented with 25 mM imidazole. The fusion protein was eluted using activity buffer with 150 mM imidazole. After 10 days at room temperature, there was no detectable spontaneous scission of G2E6 from the fused thioredoxin, in contrast to a previous report (16). The protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Arylesterase and Paraoxonase Activity Assays. To test for arylesterase activity, enzymes were incubated in reaction buffer [50 mM Tris-HCl and 10 mM CaCl2 (pH 7.4)] with increasing concentrations of phenyl acetate (Sigma-Aldrich) FIGURE 1: Structure of G2E6 PON1. (A) Residues that differ between human and G2E6 PON1 are rendered as green sticks. The 59 amino acid differences are scattered throughout the sequence, mainly on the surface of the enzyme and not in the presumed active site. Residues with atoms within 5 Å of the phosphate ion in the crystal are rendered as pink sticks. The purple spheres represent the calcium (Ca2þ) ions. (B) Residues that differ betweenG3C9 andG2E6PON1 are rendered as green sticks, whereas residues with atomswithin 5 Å of the phosphate ion in the crystal are rendered as pink sticks, as in panel A. (C)Resid