Direct detection of stereospecific soman hydrolysis by wild-type human serum paraoxonase

Human serum paraoxonase 1 (HuPON1; EC 3.1.8.1) is a calcium-dependent six-fold beta-propeller enzyme that has been shown to hydrolyze an array of substrates, including organophosphorus (OP) chemical warfare nerve agents. Although recent efforts utilizing site-directed mutagenesis have demonstrated specific residues (such as Phe222 and His115) to be important in determining the specificity of OP substrate binding and hydrolysis, little effort has focused on the substrate stereospecificity of the enzyme; different stereoisomers of OPs can differ in their toxicity by several orders of magnitude. For example, the C+/-P- isomers of the chemical warfare agent soman (GD) are known to be more toxic by three orders of magnitude. In this study, the catalytic activity of HuPON1 towards each of the four chiral isomers of GD was measured simultaneously via chiral GC/MS. The catalytic efficiency (k(cat)/K(m)) of the wild-type enzyme for the various stereoisomers was determined by a simultaneous solution of hydrolysis kinetics for each isomer. Derived k(cat)/K(m) values ranged from 625 to 4130 mm(-1).min(-1), with isomers being hydrolyzed in the order of preference C+P+ > C-P+ > C+P- > C-P-. The results indicate that HuPON1 hydrolysis of GD is stereoselective; substrate stereospecificity should be considered in future efforts to enhance the OPase activity of this and other candidate bioscavenger enzymes.     

VX hydrolysis by human serum paraoxonase 1: a comparison of experimental and computational results

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.     

Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase

Phosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme that hydrolyzes a variety of organophosphorus compounds. The kinetic parameters of Zn/Zn PTE, Cd/Cd PTE, and a mixed-metal Zn/Cd hybrid PTE were obtained with a variety of substrates to determine the role of each metal ion in binding and catalysis. pH-rate profiles for the hydrolysis of diethyl p-nitrophenyl phosphate (I) and diethyl p-chlorophenyl phosphate (II) demonstrated that the ionization of a single group in the pH range of 5-10 was critical for substrate turnover. The pK(a) values determined from the kinetic assays were dependent on the identity of the metal ion that occupied the alpha site within the binuclear metal center. These results suggest that the hydrolytic nucleophile is activated as a hydroxide via the ionization of a water molecule attached to the alpha-metal ion. The kinetic constants for the hydrolysis of II and diethyl p-chlorophenyl thiophosphate (IV) were determined for the metal substituted forms of PTE. The kinetic constants for IV were greater than those for II. The inverse thio effect is consistent with the polarization of the phosphoryl oxygen/sulfur bond via a direct ligation to the metal center. The rate enhancement is greater when Cd(2+) occupies the beta-metal-ion position. A series of alanine and asparagine mutations were used to characterize the catalytic roles of Asp233, His254, and Asp301. Mutations to either Asp233 or His254 resulted in an enhanced rate of hydrolysis for the sluggish substrate, diethyl p-chlorophenyl phosphate, and a decrease in the kinetic constants for paraoxon (I). These results are consistent with the existence of a proton relay from Asp301 to His254 to Asp233 that is used to ferry protons away from the active site with substrates that do not require activation of the leaving group phenol. A mechanism for the hydrolysis of organophosphates by the bacterial PTE has been proposed.     

Nickel(II) chelatase variants directly evolved from murine ferrochelatase: porphyrin distortion and kinetic mechanism

The heme biosynthetic pathway culminates with the ferrochelatase-catalyzed ferrous iron chelation into protoporphyrin IX to form protoheme. The catalytic mechanism of ferrochelatase has been proposed to involve the stabilization of a nonplanar porphyrin to present the pyrrole nitrogens to the metal ion substrate. Previously, we hypothesized that the ferrochelatase-induced nonplanar distortions of the porphyrin substrate impose selectivity for the divalent metal ion incorporated into the porphyrin ring and facilitate the release of the metalated porphyrin through its reduced affinity for the enzyme. Using resonance Raman spectroscopy, the structural properties of porphyrins bound to the active site of directly evolved Ni(2+)-chelatase variants are now examined with regard to the mode and extent of porphyrin deformation and related to the catalytic properties of the enzymes. The Ni(2+)-chelatase variants (S143T, F323L, and S143T/F323L), which were directly evolved to exhibit an enhanced Ni(2+)-chelatase activity over that of the parent wild-type ferrochelatase, induced a weaker saddling deformation of the porphyrin substrate. Steady-state kinetic parameters of the evolved variants for Ni(2+)- and Fe(2+)-chelatase activities increased compared to those of wild-type ferrochelatase. In particular, the reduced porphyrin saddling deformation correlated with increased catalytic efficiency toward the metal ion substrate (Ni(2+) or Fe(2+)). The results lead us to propose that the decrease in the induced protoporphyrin IX saddling mode is associated with a less stringent metal ion preference by ferrochelatase and a slower porphyrin chelation step.     

Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy

The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Up to now, the determination of kinetic data has been achieved by techniques such as pH-stat titration, ion-selective electrodes, and fluorogenic substrate analogs. We report a new assaying method using in situ Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR) for the real-time determination of reaction rates. The method employs changes in the P-O-R stretching vibration of DFP and nerve agent substrates when hydrolyzed to their corresponding phosphoric and phosphonic acids. It is shown that the Lambert-Beer law holds and that changes in absorbance can be directly related to changes in concentration. Compared with other methods, the use of in situ FTIR spectroscopy results in a substantially reduced reaction volume that adds extra work safety when handling highly toxic substrates. In addition, the new method allows the noninvasive measurement of buffered solutions with varying ionic strengths complementing existing methods. Because the assay is independent of the used enzyme, it should also be applicable to other phosphotriesterase enzymes such as organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA), and paraoxonase (PON).     

Efficient hydrolysis of the chemical warfare nerve agent tabun by recombinant and purified human and rabbit serum paraoxonase 1

Paraoxonase 1 (PON1) has been described as an efficient catalytic bioscavenger due to its ability to hydrolyze organophosphates (OPs) and chemical warfare nerve agents (CWNAs). It is the future most promising candidate as prophylactic medical countermeasure against highly toxic OPs and CWNAs. Most of the studies conducted so far have been focused on the hydrolyzing potential of PON1 against nerve agents, sarin, soman, and VX. Here, we investigated the hydrolysis of tabun by PON1 with the objective of comparing the hydrolysis potential of human and rabbit serum purified and recombinant human PON1. The hydrolysis potential of PON1 against tabun, sarin, and soman was evaluated by using an acetylcholinesterase (AChE) back-titration Ellman method. Efficient hydrolysis of tabun (100 nM) was observed with ∼25-40 mU of PON1, while higher concentration (80-250 mU) of the enzyme was required for the complete hydrolysis of sarin (11 nM) and soman (3 nM). Our data indicate that tabun hydrolysis with PON1 was ∼30-60 times and ∼200-260 times more efficient than that with sarin and soman, respectively. Moreover, the catalytic activity of PON1 varies from source to source, which also reflects their efficiency of hydrolyzing different types of nerve agents. Thus, efficient hydrolysis of tabun by PON1 suggests its promising potential as a prophylactic treatment against tabun exposure.     

In vitro stereoselective hydrolysis of diacylglycerols by hormone-sensitive lipase

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids in adipocytes and shows a substrate preference for the diacylglycerols (DAGs) originating from triacylglycerols. To determine whether HSL shows any stereopreference during the hydrolysis of diacylglycerols, racemic 1,2(2,3)-sn-diolein was used as a substrate and the enantiomeric excess (ee%) of residual 1,2-sn-diolein over 2,3-sn-diolein was measured as a function of DAG hydrolysis. Enantiomeric DAGs were separated by performing chiral-stationary-phase HPLC after direct derivatization from lipolysis product extracts. The fact that the ee% of 1,2-sn-diolein over 2,3-sn-diolein increased with the level of hydrolysis indicated that HSL has a preference for 2,3-sn-diolein as a substrate and therefore a stereopreference for the sn-3 position of dioleoylglycerol. The ee% of 1,2-sn-diolein reached a maximum value of 36% at 42% hydrolysis. Among the various mammalian lipases tested so far, HSL is the only lipolytic carboxylester hydrolase found to have a pronounced stereospecificity for the sn-3 position of dioleoylglycerol.     

Investigation of the common paraoxonase 1 variants with paraoxonase activity on bone fragility in Turkish patients

There is increasing evidence of a biochemical link between oxidative stress and bone metabolism. Oxidative stress has been shown to be involved in bone resorption as it causes loss of bone mineral density (BMD). Paraoxonase 1 (PON1), can prevent these effects of the oxidative stress on bone formation. It has been suggested that the PON1 gene as possibly implicated in reduced BMD in bone fragility cases. It has been hypothesized that PON1 gene polymorphisms may influence both the risk of osteoporosis and osteopenia occurrence and prognosis. The aim of our study is to evaluate the relationship between PON1 polymorphisms and bone fragility development. Seventy-four osteoporotic, 121 osteopenic and 79 nonosteoporotic postmenopausal women were recruited. For detection of the polymorphisms, polymerase chain reaction-restriction fragment length polymorphism techniques have been used. BMD was measured at the lumbar spine and hip by dual-energy X-ray absorptiometry. Distributions of PON1 (PON 192 and PON 55) polymorphisms in study groups were not significantly different. But, there was medium strength connection between in the osteopenic with control groups regarding PON1 55–PON1 192 haplotypes and we found a power strength connection between in the osteoporosis with control groups regarding PON1 55–PON1 192 haplotypes. Furthermore, subjects with PON1 192RR and PON1 55LL genotypes had lower PON activity values of osteoporotic subject compared to healthy control and this difference was statistically significant (p < 0.05). This result suggest that PON1 genotypes could be higher risk for osteoporosis, as determined by reduced BMD.     

Enhanced hydrolysis of cellulose hydrogels by morphological modification

Bioprocess and Biosystems Engineering - Cellulose is one of the most abundant bio-renewable materials on earth, yet the potential of cellulosic bio-fuels is not fully exploited, primarily due to...     

Enhanced enzymatic hydrolysis of sugarcane bagasse by N-methylmorpholine-N-oxide pretreatment

The cellulose dissolution solvent used in Lyocell process for cellulose fiber preparation, N-methylmorpholine-N-oxide (NMMO) monohydrate, was demonstrated to be an effective agent for sugarcane bagasse pretreatment. Bagasse of 20wt% was readily dissolved in NMMO monohydrate at 130 degrees C within 1h. After dissolution, bagasse could be regenerated by rapid precipitation with water as a porous and amorphous mixture of its original components. The regenerated bagasse exhibited a significant enhancement on enzymatic hydrolysis kinetic. Not only the reducing sugars releasing rate but also hydrolysis yield was enhanced at least twofold as compared with that of untreated bagasse. The cellulose fraction of regenerated bagasse was nearly hydrolyzed to glucose after 72h hydrolysis with Cellulase AP3. The recycled NMMO demonstrated the same performance as the fresh one on bagasse pretreatment for hydrolysis enhancement. The regenerated bagasse was directly used in simultaneous saccharification and fermentation (SSF) for ethanol production by Zymomonas mobilis. No negative effect on ethanol fermentation was observed and ethanol yield approximately 0.15 g ethanol/g baggasse was achieved.     

Engineering Pichia pastoris for stereoselective nitrile hydrolysis by co-producing three heterologous proteins

A Pichia pastoris strain with stereoselective nitrile hydratase activity has been constructed by engineering the co-expression of three genes derived from Pseudomonas putida. Using a technique that could be widely applicable, the genes encoding nitrile hydratase α and β structural subunits and P14K accessory protein were first assembled as individual expression cassettes and then incorporated onto one plasmid, which was integrated into the P. pastoris chromosome. The resulting strain can be used as a catalyst for bioconversions requiring stereospecific nitrile hydrolysis. Received: 3 November 1998 / Received revision: 25 February1999 / Accepted: 14 March 1999     

Enhanced dewatering of waste-activated sludge by composite hydrolysis enzymes

The feasibility of composite hydrolysis enzymes in enhanced dewatering of waste-activated sludge (WAS) was verified in this study. A Pearson correlation analysis was conducted to explore the roles of different extracellular polymeric substance (EPS) fractions on WAS dewaterability. The results indicated that tightly bound EPS (TB-EPS) was released into the liquid phase consistently during enzymatic hydrolysis to form soluble EPS (S-EPS) and loosely bound EPS and that the TB-EPS content was positively correlated with the capillary suction time of WAS. A kinetic analysis was carried out to gain further insights into the kinetic variation in TB-EPS removal. It was found that TB-EPS reduction fit a first-order kinetic model and that mild temperature (25–30 °C) and a slightly acidic condition were favorable for the improvement of enzyme activity. Solid phase extraction combined with UV–Vis spectroscopy analysis was used to characterize the processes of migration and transformation of the hydrophobic (HPO), transphilic and hydrophilic (HPI) fractions in EPS during the enzymatic process. The results revealed that HPO and HPI were mainly composed of PN and PS, respectively, and that the enzymatic hydrolysis could enhance the transformation of HPI from TB-EPS to S-EPS, which was the dominant mechanism of improving WAS dewaterability.     

Enkephalin hydrolysis by human serum biotinidase.

Purified human serum biotinidase exhibited amino-exo-peptidase activity. Enkephalins and dynorphin A (less than 10-mer) seemed to be the most appropriate substrates among various physiological peptides in terms of the kcat/Km values. Similar kcat/Km values were obtained for both biocytin (biotinyllysine) and these opioid-neuropeptides. Neuro-oligo-peptides ranging from 2-mer to 18-mer were hydrolyzed. The presence of amino group at the carboxyl terminal position increased the kcat/Km value by decreasing the Km value. The results of inhibition studies using various kinds of antibiotic inhibitors, metals, and chelating agents indicated that enkephalin hydrolysis was mediated by the peptide-hydrolyzing center probably containing Zn ions. This aminopeptidase activity was uniquely inhibited by a vitamin of biocytin. The reason for the high content of biotinidase activity in serum may be related to the binary function of this enzyme; i.e., biocytin hydrolyzing amidase and enkephalin hydrolyzing aminopeptidase functions.     

Determinants of variation in human serum paraoxonase activity

Paraoxonase-1 (PON1) is associated with high-density lipoprotein (HDL) particles and is believed to contribute to antiatherogenic properties of HDLs. We assessed the determinants of PON1 activity variation using different substrates of the enzyme. PON1 activity in serum samples from 922 participants in the San Antonio Family Heart Study was assayed using a reliable microplate format with three substrates: paraoxon, phenyl acetate and the lactone dihydrocoumarin. There were major differences among results from the three substrates in degree of effect by various environmental and genetic factors, suggesting that knowledge of one substrate activity alone may not provide a complete sense of PON1 metabolism. Three significant demographic covariates (age, smoking status and contraceptive usage) together explained 1-6% of phenotypic variance, whereas four metabolic covariates representing lipoprotein metabolism (apoAII, apoAI, triglycerides and non-HDL cholesterol) explained 4-19%. Genes explained 65-92% of phenotypic variance and the dominant genetic effect was exerted by a locus mapping at or near the protein structural locus (PON1) on chromosome 7. Additional genes influencing PON1 activity were localized to chromosomes 3 and 14. Our study identified environmental and genetic determinants of PON1 activity that accounted for 88-97% of total phenotypic variance, suggesting that few, if any, major biological determinants are unrepresented in the models.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

Enhanced enzymatic hydrolysis of wheat straw by aqueous glycerol pretreatment

Considering the practical technology-economy of glycerol processing from oleochemicals industry, the ensuing work was proposed to further explore the atmospheric aqueous glycerol autocatalytic organosolv pretreatment (AAGAOP) to improve the enzymatic hydrolysis of lignocellulosic biomass. With the liquid-solid ratio of 20 g g(-1) at 220 degrees C for 3h, the AAGAOP enabled wheat straw to remove approximately 70% hemicelluloses and approximately 65% lignin, with approximately 98% cellulose retention. The pretreated fiber was achieved with approximately 90% of the enzymatic hydrolysis yield after 48 h. At oven-drying, dehydration was likely to cause the hornification of fiber, which was responsible for the low enzymatic hydrolysis of dried fiber. With SEM observations, the AAGAOP disrupted wheat straw into thin and fine fibrils, with a small average size and more surface area. The AAGAOP technique, as a novel strategy, enhanced the enzymatic hydrolysis of lignocellulosic biomass by removing the chemically compositional barrier and altering the physically structural impediment.     

Effect of serum lipoproteins on stereoselective halofantrine metabolism by rat hepatocytes

Experimental hyperlipidemia has shown to decrease cytochrome P450 3A4 and 2C11 expression and to increase liver concentrations and the plasma protein binding of halofantrine (HF) enantiomers. The present study examined the effect of hyperlipidemic (HL) serum on the metabolism of HF enantiomers by primary rat hepatocytes. Hepatocytes from normolipidemic (NL) and HL (poloxamer 407 treated) rats were incubated with rac-HF in cell media with or without additional rat serum (5%). In those incubations with rat serum, the hepatocytes were preincubated or coincubated with serum from NL or HL rats. Rat serum-free hepatocyte incubations served as controls. Stereospecific assays were used to measure HF and desbutylhalofantrine (its major metabolite) enantiomer concentrations in whole well contents (cells + media). Concentrations of desbutylhalofantrine were not measurable. The disappearance (apparent metabolism) of (-)-HF exceeded that of antipode, but HF metabolism did not differ between hepatocytes from NL and HL rats. Coincubation of HL rat serum with NL hepatocytes caused a significant decrease in the disappearance of (-)-HF, whereas in HL hepatocytes, a substantially decreased apparent metabolism was noted for both enantiomers. Compared with NL serum, (-)-HF disappearance was significantly lowered upon preincubation of NL hepatocytes with HL serum. A combination of factors including diminished drug metabolizing or lipoprotein receptor expression, and increased plasma protein binding in the wells, may have contributed to a decrease in apparent metabolism of the HF enantiomers in the presence of lipoproteins from HL rat serum.