Biomedical subjects

R Wever

Publications and source records attributed to R Wever.

At least 19 recordsLinked to original sources

PubMed2001

Determination of the toxicity of several aromatic carbonylic compounds and their reduced derivatives on Phanerochaete chrysosporium using a Pseudomonas putida test system.

We tested four aromatic carbonylic compounds and their corresponding reduced derivatives, possible substrates, and products of a biotransformation for toxicity against the white-rot fungus Phanerochaete chrysosporium. The bacterium Pseudomonas putida, which has been proven to be a good test organism for investigating toxic effects, was used as a primary screen. For both P. chrysosporium and P. putida, all ketones showed a higher toxicity than their corresponding alcohol derivatives. Within one chemical group a direct correlation between the hydrophobicity (logP values) of the compounds and their toxicity could be observed. Furthermore, all tested compounds also caused an isomerization of cis to trans unsaturated fatty acids in P. putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences. Toxicity of aromatic carbonylic compounds in an established biotransformation system with P. chrysosporium can be estimated by calculating the corresponding logP values of the substrates and potential products. P. putida can be used to test the toxicity of aromatic ketones to the basic diomycete P. chrysosporium.

Alcohols↗

PubMed2001

Vanadium haloperoxidases from brown algae of the Laminariaceae family.

Vanadium haloperoxidases were extracted, purified and characterized from three different species of Laminariaceae--Laminaria saccharina (Linné) Lamouroux, Laminaria hyperborea (Gunner) Foslie and Laminaria ochroleuca de la Pylaie. Two different forms of the vanadium haloperoxidases were purified from L. saccharina and L. hyperborea and one form from L. ochroleuca species. Reconstitution experiments in the presence of several metal ions showed that only vanadium(V) completely restored the enzymes activity. The stability of some enzymes in mixtures of buffer solution and several organic solvents such as acetone, ethanol, methanol and 1-propanol was noteworthy; for instance, after 30 days at least 40% of the initial activity for some isoforms remained in mixtures of 3:1 buffer solution/organic solvent. The enzymes were also moderately thermostable, keeping full activity up to 40 degrees C. Some preliminary steady-state kinetic studies were performed and apparent Michaelis-Menten kinetic parameters were determined for the substrates iodide and hydrogen peroxide. Histochemical studies were also performed in fresh tissue sections from stipe and blade of L. hyperborea and L. saccharina, showing that haloperoxidase activity was concentrated in the external cortex near the cuticle, although some activity was also observed in the inner cortical region.

Chromatography, Ion Exchange↗

PubMed2001

Sulfoxidation mechanism of vanadium bromoperoxidase from Ascophyllum nodosum. Evidence for direct oxygen transfer catalysis.

We have previously shown that vanadium bromoperoxidase from Ascophyllum nodosum mediates production of the (R)-enantiomer of methyl phenyl sulfoxide with 91% enantiomeric excess. Investigation of the intrinsic selectivity of vanadium bromoperoxidase reveals that the enzyme catalyzes the sulfoxidation of methyl phenyl sulfide in a purely enantioselective manner. The K(m) of the enzyme for methyl phenyl sulfide was determined to be approximately 3.5 mM in the presence of 25% methanol or tert-butanol. The selectivity of the sulfoxidation of methyl phenyl sulfide is optimal in the temperature range 25-30 degrees C and can be further optimized by increasing the enzyme concentration, yielding selectivities with up to 96% enantiomeric excess. Furthermore, we established for the first time that vanadium bromoperoxidase is functional at temperatures up to 70 degrees C. A detailed investigation of the sulfoxidation activity of this enzyme using (18)O-labeled hydrogen peroxide shows that vanadium bromoperoxidase mediates the direct transfer of the peroxide oxygen to the sulfide. A schematic model of the vanadium haloperoxidase sulfoxidation mechanism is presented.

Catalysis↗

PubMed2000

Oxidation reactions catalyzed by vanadium chloroperoxidase from Curvularia inaequalis.

Vanadium haloperoxidases have been reported to mediate the oxidation of halides to hypohalous acid and the sulfoxidation of organic sulfides to the corresponding sulfoxides in the presence of hydrogen peroxide. However, traditional heme peroxidase substrates were reported not to be oxidized by vanadium haloperoxidases. Surprisingly, we have now found that the recombinant vanadium chloroperoxidase from the fungus Curvularia inaequalis catalyzes the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), a classical chromogenic heme peroxidase substrate. The enzyme mediates the oxidation of ABTS in the presence of hydrogen peroxide with a turnover frequency of 11 s(-1) at its pH optimum of 4.0. The Km of the recombinant enzyme for ABTS was observed to be approximately 35 microM at this pH value. In addition, the bleaching of an industrial sulfonated azo dye, Chicago Sky Blue 6B, catalyzed by the recombinant vanadium chloroperoxidase in the presence of hydrogen peroxide is reported.

Chloride Peroxidase↗

PubMed2000

Peroxidase and phosphatase activity of active-site mutants of vanadium chloroperoxidase from the fungus Curvularia inaequalis. Implications for the catalytic mechanisms.

Mutation studies were performed on active-site residues of vanadium chloroperoxidase from the fungus Curvularia inaequalis, an enzyme which exhibits both haloperoxidase and phosphatase activity and is related to glucose-6-phosphatase. The effects of mutation to alanine on haloperoxidase activity were studied for the proposed catalytic residue His-404 and for residue Asp-292, which is located close to the vanadate cofactor. The mutants were strongly impaired in their ability to oxidize chloride but still oxidized bromide, although they inactivate during turnover. The effects on the optical absorption spectrum of vanadium chloroperoxidase indicate that mutant H404A has a reduced affinity for the cofactor, whereas this affinity is unchanged in mutant D292A. The effect on the phosphatase activity of the apoenzyme was investigated for six mutants of putative catalytic residues. Effects of mutation of His-496, Arg-490, Arg-360, Lys-353, and His-404 to alanine are in line with their proposed roles in nucleophilic attack, transition-state stabilization, and leaving-group protonation. Asp-292 is excluded as the group that protonates the leaving group. A model based on the mutagenesis studies is presented and may serve as a template for glucose-6-phosphatase and other related phosphatases. Hydrolysis of a phospho-histidine intermediate is the rate-determining step in the phosphatase activity of apochloroperoxidase, as shown by burst kinetics.

Binding Sites↗

PubMed2000

Cofactor and substrate binding to vanadium chloroperoxidase determined by UV-VIS spectroscopy and evidence for high affinity for pervanadate.

The vanadate cofactor in vanadium chloroperoxidase has been studied using UV-VIS absorption spectroscopy. A band is present in the near-UV that is red-shifted as compared to free vanadate and shifts in both position and intensity upon change in pH. Mutation of vanadate binding residues has a clear effect on the spectrum. Substrate-induced spectral effects allow direct measurement of separate kinetics steps for the first time for vanadium haloperoxidases. A peroxo intermediate is formed upon addition of H(2)O(2), which causes a decrease in the absorption spectrum at 315 nm, as well as an increase at 384 nm. This peroxo form is very stable at pH 8.3, whereas it is less stable at pH 5.0, which is the optimal pH for activity. Upon addition of halides to the peroxo form, the native spectrum is re-formed as a result of halide oxidation. Stopped-flow experiments show that H(2)O(2) binding and Cl(-) oxidation occur on the millisecond to second time scale. These data suggest that the oxidation of Cl(-) to HOCl occurs in at least two steps. In the presence of H(2)O(2), the affinity for the vanadate cofactor was found to be much higher than previously reported for vanadate in the absence of H(2)O(2). This is attributed to the uptake of pervanadate by the apo-enzyme. Human glucose-6-phosphatase, which is evolutionarily related to vanadium chloroperoxidase, is also likely to have a higher affinity for pervanadate than vanadate. This could explain the enhanced insulin mimetic effect of pervanadate as compared to vanadate.

Apoenzymes↗

PubMed2000

Enantioselective epoxidation and carbon-carbon bond cleavage catalyzed by Coprinus cinereus peroxidase and myeloperoxidase.

We demonstrate that myeloperoxidase (MPO) and Coprinus cinereus peroxidase (CiP) catalyze the enantioselective epoxidation of styrene and a number of substituted derivatives with a reasonable enantiomeric excess (up to 80%) and in a moderate yield. Three major differences with respect to the chloroperoxidase from Caldariomyces fumago (CPO) are observed in the reactivity of MPO and CiP toward styrene derivatives. First, in contrast to CPO, MPO and CiP produced the (S)-isomers of the epoxides in enantiomeric excess. Second, for MPO and CiP the H(2)O(2) had to be added very slowly (10 eq in 16 h) to prevent accumulation of catalytically inactive enzyme intermediates. Under these conditions, CPO hardly showed any epoxidizing activity; only with a high influx of H(2)O(2) (300 eq in 1.6 h) was epoxidation observed. Third, both MPO and CiP formed significant amounts of (substituted) benzaldehydes as side products as a consequence of C-alpha-C-beta bond cleavage of the styrene derivatives, whereas for CPO and cytochrome c peroxidase this activity is not observed. C-alpha-C-beta cleavage was the most prominent reaction catalyzed by CiP, whereas with MPO the relative amount of epoxide formed was higher. This is the first report of peroxidases catalyzing both epoxidation reactions and carbon-carbon bond cleavage. The results are discussed in terms of mechanisms involving ferryl oxygen transfer and electron transfer, respectively.

Carbon↗

PubMed2000

Purification and characterisation of vanadium haloperoxidases from the brown alga Pelvetia canaliculata.

Two enzymes characterised as iodoperoxidases (PcI and PcII), with vanadium-dependent activity, have been purified from the brown alga Pelvetia canaliculata (L.) Decne et Thur. (Fucaceae, Phaeophyceae), collected in the Northern Portuguese coast, at Viana do Castelo. The relative molecular masses were 166 kDa for PcI and 416 kDa for PcII, as determined by gel filtration. SDS-PAGE shows that PcI has just one band corresponding to a subunit of 66 kDa, while PcII shows four bands (66, 72, 157 and 280 kDa). The following kinetic parameters have been determined from a steady-state analysis of the oxidation of iodide by H2O2: PcI, pHopt = 6.0, KM(I-) = 2.1 mM, KM(H2O2) = 110 microM, Ki(I-) = 127 mM; and PcII, pHopt = 6.5, KM(I-) = 2.4 mM, KM(H2O2) = 20 microM and Ki(I-) = 69 mM. These iodoperoxidases are thermostable, as also observed for vanadium bromo- and chloroperoxidases.

Catalytic Domain↗

PubMed2000

Influence of folic acid on postprandial endothelial dysfunction.

Triglyceride-rich lipoproteins that circulate postprandially are increasingly being recognized as potentially atherogenic. These particles also have been shown to cause endothelial dysfunction. We recently demonstrated that acute parenteral administration of folic acid restores endothelial function in vivo in patients with increased LDL cholesterol levels. In vitro data suggested that this effect could be mediated by a reduction of radical stress. In the present study, therefore, we evaluated the effect of an acute oral fat load on both endothelial function and oxygen radical production. Next, we studied whether 2 weeks of pretreatment with 10 mg folic acid PO could prevent these fat-induced changes. We conducted a prospective, randomized, placebo-controlled study to evaluate the effect of oral folic acid administration (10 mg/d for 2 weeks) on basal endothelial function as well as endothelial function on an acute fat load in 20 healthy volunteers 18 to 33 years old. Endothelial function was assessed as flow-mediated dilatation (FMD). Endothelium-independent dilatation was measured after sublingual nitroglycerin spray. Oxygen radical stress was assessed by measurement of the urinary excretion of the stable radical-damage end product malondialdehyde. During administration of placebo, FMD decreased significantly after an acute oral fat load, with a median from 10.6% (8.3% to 12.2%) to 5.8% (3.0% to 10.2%), P<0.05. During folic acid administration, FMD was unaffected by a fat load, with a median from 9.6% (7.1% to 12.8%) to 9.9% (7.5% to 14.1%), P=NS. The increase in malondialdehyde excretion in the urine after fat loading was also prevented during folic acid administration (absolute increase after an acute fat load during placebo, 0.11+/-0.1 micromol/L versus folic acid, 0.02+/-0.1 micromol/L, P<0.05). The response to the endothelium-independent vasodilator nitroglycerin remained unaltered throughout the study. Pretreatment with oral folic acid prevents the lipid-induced decrease in FMD as well as the lipid-induced increase in urinary radical-damage end products. Because these observations were made in healthy volunteers with normal folate and homocysteine levels, it is suggested that a higher folate intake in the general population may have vasculoprotective effects.

Adolescent↗

PubMed1999

The sulfonium ion linkage in myeloperoxidase. Direct spectroscopic detection by isotopic labeling and effect of mutation.

The heme group of myeloperoxidase is covalently linked via two ester bonds to the protein and a unique sulfonium ion linkage involving Met(243). Mutation of Met(243) into Thr, Gln, and Val, which are the corresponding residues of eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase, respectively, and into Cys was performed. The Soret band in the optical absorbance spectrum in the oxidized mutants is now found at approximately 411 nm. Both the pyridine hemochrome spectra and resonance Raman spectra of the mutants are affected by the mutation. In the Met(243) mutants the affinity for chloride has decreased 100-fold. All mutants have lost their chlorination activity, except for the M243T mutant, which still has 15% activity left. By Fourier transform infared difference spectroscopy it was possible to specifically detect the (13)CD(3)-labeled methionyl sulfonium ion linkage. We conclude that the sulfonium ion linkage serves two roles. First, it serves as an electron-withdrawing substituent via its positive charge, and, second, together with its neighboring residue Glu(242), it appears to be responsible for the lower symmetry of the heme group and distortion from the planar conformation normally seen in heme-containing proteins.

Amino Acid Substitution↗

PubMed1999

Heterologous expression of the vanadium-containing chloroperoxidase from Curvularia inaequalis in Saccharomyces cerevisiae and site-directed mutagenesis of the active site residues His(496), Lys(353), Arg(360), and Arg(490).

The vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis is heterologously expressed to high levels in the yeast Saccharomyces cerevisiae. Characterization of the recombinant enzyme reveals that this behaves very similar to the native chloroperoxidase. Site-directed mutagenesis is performed on four highly conserved active site residues to examine their role in catalysis. When the vanadate-binding residue His(496) is changed into an alanine, the mutant enzyme loses the ability to bind vanadate covalently resulting in an inactive enzyme. The negative charges on the vanadate oxygens are compensated by hydrogen bonds with the residues Arg(360), Arg(490), and Lys(353). When these residues are changed into alanines the mutant enzymes lose the ability to effectively oxidize chloride but can still function as bromoperoxidases. A general mechanism for haloperoxidase catalysis is proposed that also correlates the kinetic properties of the mutants with the charge and the hydrogen-bonding network in the vanadate-binding site.

Catalytic Domain↗

PubMed1999

X-ray crystal structures of active site mutants of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis.

The X-ray structures of the chloroperoxidase from Curvularia inaequalis, heterologously expressed in Saccharomyces cerevisiae, have been determined both in its apo and in its holo forms at 1.66 and 2.11 A resolution, respectively. The crystal structures reveal that the overall structure of this enzyme remains nearly unaltered, particularly at the metal binding site. At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interactions and hydrogen bonds with positively charged residues involved in the interactions with the vanadate in the native protein. The vanadate binding pocket seems to form a very rigid frame stabilising oxyanion binding. The rigidity of this active site matrix is the result of a large number of hydrogen bonding interactions involving side chains and the main chain of residues lining the active site. The structures of single site mutants to alanine of the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed. Additionally we determined the structural effects of mutations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involved in forming a salt bridge with Arg490 which also interacts with the vanadate. The enzymatic chlorinating activity is drastically reduced to approximately 1% in mutants D292A, H404A and H496A. The structures of the mutants confirm the view of the active site of this chloroperoxidase as a rigid matrix providing an oxyanion binding site. No large changes are observed at the active site for any of the analysed mutants. The empty space left by replacement of large side chains by alanines is usually occupied by a new solvent molecule which partially replaces the hydrogen bonding interactions to the vanadate. The new solvent molecules additionally replace part of the interactions the mutated side chains were making to other residues lining the active site frame. When this is not possible, another side chain in the proximity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame.

Binding Sites↗

PubMed1999

The Met243 sulfonium ion linkage is responsible for the anomalous magnetic circular dichroism and optical spectral properties of myeloperoxidase.

The heme group of myeloperoxidase shows anomalous optical properties, and the enzyme possesses the unique ability to catalyze the oxidation of chloride. However, the nature of the covalently bound heme macrocycle has been difficult to identify. In this work, the electronic and magnetic properties of the heme groups in oxidized and reduced forms of wild-type and Met243Thr mutant myeloperoxidase and wild-type lactoperoxidase have been investigated using variable-temperature (1.6-273 K) magnetic circular dichroism (MCD) spectroscopy along with parallel optical absorption and electron paramagnetic resonance studies. The results provide assessment of the spin state mixtures of the oxidized and reduced samples at ambient and liquid helium temperatures and show that the anomalous MCD properties of myeloperoxidase, e.g. red-shifted and inverted signs for bands in the high-spin ferric and low-spin ferrous forms compared to other heme peroxidases and heme proteins in general, are a direct consequence of a novel electron-withdrawing sulfonium ion heme linkage involving Met243.

Circular Dichroism↗

PubMed1999

Characterization of the Asp94 and Glu242 mutants in myeloperoxidase, the residues linking the heme group via ester bonds.

The heme group of all mammalian peroxidases is covalently linked to the protein matrix via two esterbonds, as we have recently shown by Fourier transform infrared (FTIR) difference spectroscopy [Kooter, I. M., Pierik, A.J., Merkx, M., Averill, B.A., Moguilevsky, N., Bollen, A. & Wever, R. (1997) J. Am. Chem. Soc. 119, 11542-11543]. We have examined the effects of mutation of Asp94 and Glu242, responsible for those ester bonds in myeloperoxidase, on the spectroscopic properties and catalytic activity of this enzyme. Mutation of Asp94 in myeloperoxidase results in two species. The first species has spectroscopic characteristics similar to that of wild-type myeloperoxidase. The second species has spectroscopic characteristics similar to that of Met243-->Gln mutant, and it is therefore concluded that, besides loss of the ester bond involving Asp94, this species also has lost the sulfonium ion linkage that is also characteristic of myeloperoxidase. The Asp94-->Asn mutant still has about 30% residual peroxidase activity while for the Asp94-->Val mutant only a few percentage activity is left. When Glu242 is mutated the sulfonium ion linkage is not affected, but this residue together with its neighbouring residue Met243, according to resonance Raman spectra, is responsible for the low symmetry of the heme group. Mutation of either of these residues results in loss of the bowed distortion from the planar conformation, and in a heme group with higher symmetry. For the Glu242-->Gln mutant 8% residual peroxidase activity is found.

Aspartic Acid↗

PubMed1999

Nitric oxide production is reduced in patients with chronic renal failure.

In patients with chronic renal failure (CRF), atherosclerosis is a major cause of cardiovascular morbidity and mortality. Generally, atherosclerosis has been associated with a reduced bioavailability of nitric oxide (NO). Experimental studies have indicated the presence of enhanced NO degradation by reactive oxygen species as well as decreased NO production as possible causes for this reduced NO bioavailability. So far, the question whether or not NO production is impaired in patients with CRF has never been investigated. Therefore, we measured whole body NO production in 7 patients with CRF, and in 7 matched healthy subjects. To assess the relative importance of a dysfunction of NO synthase (NOS), we compared the NO production of these patients to that of 2 other groups known to have endothelial dysfunction, ie, 7 patients with familial hypercholesterolemia (FH) who did not yet have signs of clinical cardiovascular disease (all nonsmokers), and 5 cigarette smokers. These groups were also compared with 7 nonsmoking, age-matched healthy subjects. Whole body NO production, determined as in vivo arginine-to-citrulline conversion, was assessed by giving an intravenous infusion of [15N2]-arginine as a substrate for NOS and measuring isotopic plasma enrichment of [15N]-citrulline by LC-MS. NO production in the CRF patients (0.13+/-0.02 micromol. kg-1. h-1) was significantly lower (P<0.05) than in the corresponding control group (0.23+/-0.09 micromol. kg-1. h-1). NO production also tended to be lower in the FH patients (0.16+/-0.04 micromol. kg-1. h-1), but the difference with the corresponding control group did not reach significance (0.22+/-0.06 micromol. kg-1. h-1). In the group of smokers, NO production was similar to that in nonsmokers (0. 22+/-0.09 micromol. kg-1. h-1). In conclusion, it is demonstrated for the first time that basal whole body NO production is reduced in patients with CRF. This finding implies that therapeutic interventions to endothelial dysfunction in these patients should be primarily directed toward improvement of NO production. The finding of only a tendency toward reduction of NO production in patients with FH and the absence of a reduction in cigarette smokers suggests that other mechanisms such as enhanced NO degradation may be involved in the decrease of NO bioavailability in these groups.

Arginine↗

PubMed1998

The sulphoxidation of thioanisole catalysed by lactoperoxidase and Coprinus cinereus peroxidase: evidence for an oxygen-rebound mechanism.

Using both stopped-flow and conventional spectroscopy, the oxygenation of methyl phenyl sulphide by both lactoperoxidase (LPO) and Coprinus cinereus peroxidase (CiP) was monitored. Controlled continuous addition of H2O2 during turnover and monitoring the presence of native enzymes, compounds I, II and III, led to formation of the sulphoxide in high yield and enantioselectivity. Under those conditions, LPO catalysed the formation of (R) methyl phenyl sulphoxide with a yield of 85% and an enantiomeric excess (e.e.) of 80%. CiP catalysed the formation of (S) methyl phenyl sulphoxide with a yield of 84% and an e.e. of 73%. The enantioselective performance was markedly influenced by the purity of the enzymes used. Presence of compound III during turnover led to rapid inactivation of the peroxidases and, therefore, to both a lower yield of the sulphoxides and a lower enantioselectivity. Stopped-flow kinetic data show that, for both LPO and CiP, the transition of compound I to compound II depends on the concentration of the methyl phenyl sulphide, suggesting an oxygen-rebound mechanism. In line with this mechanism, a methyl phenyl sulphide radical cation was detected by EPR during turnover for LPO.

Coprinus↗

PubMed1998

Origin of superoxide production by endothelial nitric oxide synthase.

Using fluorescence optical and electron spin resonance spectroscopy, we have investigated the production of superoxide by bovine endothelial nitric oxide synthase (NOS). In contrast to neuronal NOS, the heme moiety is identified as the exclusive source of superoxide production by endothelial NOS. Thus, calmodulin-mediated enzyme regulation affects production of nitric oxide and superoxide simultaneously and inseparably. The balance between the nitric oxide/superoxide reaction pathways may be shifted by addition of exogenous heme-specific agents, such as tetrahydrobiopterin. Our results have direct relevance for the pathophysiology of atherosclerosis.

Animals↗

PubMed1998

Isolation, characterization, and primary structure of the vanadium chloroperoxidase from the fungus Embellisia didymospora.

Here we describe the isolation, purification, and basic kinetic parameters of a vanadium type chloroperoxidase from the hyphomycete fungus Embellisia didymospora. The enzyme proved to possess similar high substrate affinities, a Km of 5 microM for a bromide, 1.2 mM for a chloride, and 60 microM for a hydrogen peroxide, as those of the vanadium chloroperoxidase from Curvularia inaequalis, although with lower turnover rates for both Cl- and Br-. Substrate bromide was also found to inhibit the enzyme, a feature subsequently also noted for the chloroperoxidase from C. inaequalis. The gene encoding this enzyme was identified using DNA Southern blotting techniques and subsequently isolated and sequenced. A comparison is made between this vanadium chloroperoxidase and that of the fungus C. inaequalis both kinetically and at the sequence level. At the primary structural level the two chloroperoxidases share 68% identity, with conservation of all active site residues.

Amino Acid Sequence↗