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R Wever

Publications and source records attributed to R Wever.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cloning and expression of the gene for a vanadium-dependent bromoperoxidase from a marine macro-alga, Corallina pilulifera.

The cDNAs for a vanadium-dependent bromoperoxidase were cloned from a marine macro-alga, Corallina pilulifera. The open reading frame of one clone (bpo1) encoded a protein of 598 amino acids with a calculated molecular mass of 65312 Da in good agreement with that of 64 kDa determined for the native enzyme. The deduced amino acid sequence coincided well with partial sequences of peptide fragments of the enzyme. From the same cDNA library we also isolated another cDNA clone (bpo2) encoding a protein of 597 amino acids with an identity of about 90% to BPO1, suggesting a genetic diversity of the bromoperoxidase gene of C. pilulifera growing in a relatively narrow area. The carboxy-terminal 123 residues of the enzyme (BPO1) showed an identity of 45% to that of the marine macro-alga Ascophillum nodosum. The homology search of the sequences of bromoperoxidases from C. pilulifera (this study) and A. nodostum, and chloroperoxidase from the fungus Curvularia inaequalis indicated highly conserved sequences PxYxSGHA and LxxxxAxxRxxxGxHxxxD. Furthermore, it was found that the histidine residue directly bound to vanadium, other residues building up the metal center and catalytic histidine residue forming the active site of the chloroperoxidase from C. inaequalis are conserved in the primary structure of the bromoperoxidase from C. pilulifera. The cloned hpol was introduced into Escherichia coli, and the expressed PO1 was purified from the recombinant strain. The N-terminal amino acid sequence of the purified BPO1 was identical to the deduced sequence from the cDNA except the N-terminal methionine.

Amino Acid Sequence↗
PubMed1997

A new model for the membrane topology of glucose-6-phosphatase: the enzyme involved in von Gierke disease.

Very recently we have proposed [Hemrika et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2145-2149] that the active site of the vanadate-containing chloroperoxidase from the fungus Curvularia inaequalis, of which the tertiary structure is known, is structurally very similar to that of the membrane-bound mammalian glucose-6-phosphatases for which no structural data are available. The proposed active site of glucose-6-phosphatase, however, is incompatible with the six transmembrane-helix topology model that is currently used. Here we present a new topology model for glucose-6-phosphatase which is in agreement with all available data.

Amino Acid Sequence↗
PubMed1997

The regulation of the vanadium chloroperoxidase from Curvularia inaequalis.

The effects of carbon and nitrogen source on the regulation of the vanadium chloroperoxidase secreted by the fungus Curnularia inaequalis were investigated. The addition of glucose showed a repressing effect on both the observed messenger RNA level and the measured enzyme activities, whereas the addition of glutamate as nitrogen source and the addition of both glutamate and glycerol had no effect. Addition of vanadate had no effect on the level of mRNA. Eight hundred base pairs of the upstream promoter region of vCPO were sequenced and various features of interest are highlighted. Closer inspection of the mycelium revealed that once secreted, vCPO probably remains tightly associated with the hyphae in two forms, one of which may be a proform of the enzyme. A possible cleavage event at the C-terminus may lower its potential for hyphal association and permit its disassociation into the growth medium. A putative role for the vanadium chloroperoxidase is put forward.

Base Sequence↗
PubMed1997

From phosphatases to vanadium peroxidases: a similar architecture of the active site.

We show here that the amino acid residues contributing to the active sites of the vanadate containing haloperoxidases are conserved within three families of acid phosphatases; this suggests that the active sites of these enzymes are very similar. This is confirmed by activity measurements showing that apochloroperoxidase exhibits phosphatase activity. These observations not only reveal interesting evolutionary relationships between these groups of enzymes but may also have important implications for the research on acid phosphatases, especially glucose-6-phosphatase-the enzyme affected in von Gierke disease-of which the predicted membrane topology may have to be reconsidered.

Acid Phosphatase↗
PubMed1997

Tetrahydrobiopterin restores endothelial function in hypercholesterolemia.

In hypercholesterolemia, impaired nitric oxide activity has been associated with increased nitric oxide degradation by oxygen radicals. Deficiency of tetrahydrobiopterin, an essential cofactor of nitric oxide synthase, causes both impaired nitric oxide activity and increased oxygen radical formation. In this study we tested whether tetrahydrobiopterin deficiency contributes to the decreased nitric oxide activity observed in hypercholesterolemic patients. Therefore, L-mono-methyl-arginine to inhibit basal nitric oxide activity, serotonin to stimulate nitric oxide activity, and nitroprusside as endothelium-independent vasodilator were infused in the brachial artery of 13 patients with familial hypercholesterolemia and 13 matched controls. The infusions were repeated during coinfusion of L-arginine (200 microg/kg/min), tetrahydrobiopterin (500 microg/min), or the combination of both compounds. Forearm vasomotion was assessed using forearm venous occlusion plethysmography and expressed as ratio of blood flow between measurement and control arm (M/C ratio). Tetrahydrobiopterin infusion alone did not alter M/C ratio. Both the attenuated L-mono-methyl-arginine-induced vasoconstriction as well as the impaired serotonin-induced vasodilation were restored in patients during tetrahydrobiopterin infusion. Tetrahydrobiopterin had no effect in controls. In conclusion, this study demonstrates restoration of endothelial dysfunction by tetrahydrobiopterin suppletion in hypercholesterolemic patients.

Adult↗
PubMed1996

X-ray structure of a vanadium-containing enzyme: chloroperoxidase from the fungus Curvularia inaequalis.

The chloroperoxidase (EC 1.11.1.-) from the fungus Curvularia inaequalis belongs to a class of vanadium enzymes that oxidize halides in the presence of hydrogen peroxide to the corresponding hypohalous acids. The 2.1 A crystal structure (R = 20%) of an azide chloroperoxidase complex reveals the geometry of the catalytic vanadium center. Azide coordinates directly to the metal center, resulting in a structure with azide, three nonprotein oxygens, and a histidine as ligands. In the native state vanadium will be bound as hydrogen vanadate(V) in a trigonal bipyramidal coordination with the metal coordinated to three oxygens in the equatorial plane, to the OH group at one apical position, and to the epsilon 2 nitrogen of a histidine at the other apical position. The protein fold is mainly alpha-helical with two four-helix bundles as main structural motifs and an overall structure different from other structures. The helices pack together to a compact molecule, which explains the high stability of the protein. An amino acid sequence comparison with vanadium-containing bromoperoxidase from the seaweed Ascophyllum nodosum shows high similarities in the regions of the metal binding site, with all hydrogen vanadate(V) interacting residues conserved except for lysine-353, which is an asparagine.

Amino Acid Sequence↗
PubMed1995

Primary structure and characterization of the vanadium chloroperoxidase from the fungus Curvularia inaequalis.

Using reverse transcription of messenger RNA followed by amplification using the polymerase chain reaction, three overlapping cDNA fragments encompassing the encoding sequence of the vanadium chloroperoxidase from the fungus Curvularia inaequalis were isolated and sequenced. The sequence was confirmed by DNA sequence analysis of genomic DNA. The deduced amino acid sequence predicts a protein of 609 residues with a mass of 67488 Da. Competitive reverse-transcription polymerase chain reaction analysis indicates that vanadium chloroperoxidase expression takes place in the secondary-growth phase initiated by nutrient depletion. Southern-blot analysis of genomic DNA indicates that there is only a single gene encoding the vanadium chloroperoxidase and that no isoenzymes are present. The N-terminal amino acid residue was blocked and could not be determined by amino acid sequencing, although evidence is presented showing that the N-terminal region starts very close to the first encoded methionine residue. Although the vanadium chloroperoxidase is secreted, it was not possible to assign a leader peptide. The protein contains two putative N-glycosylation sites but experiments indicate that the protein is non-glycosylated. Two cysteine residues are present in the protein both as free thiols: no disulphide bridging was found. Metal analysis revealed that iron, copper, and calcium do not constitute part of the protein. Zinc was found at a ratio of 0.3 +/- 0.04 mol/mol protein. Boiling and subsequent SDS/PAGE of the protein sample showed a typical degradation pattern of the enzyme. Amino acid sequence analysis of the resulting peptides showed that the cleavage took place at Asp-Pro bonds of which six are located throughout the protein. No sequence similarity with other known peroxidases was found except for one small region, sharing limited similarity with bacterial haloperoxidases and other alpha/beta-hydrolase-fold enzymes. In the case of the bacterial bromoperoxidases from this group, a methionine located in this region was suggested to have a role in catalysis. Methionine, however, was not involved in the catalysis of the vanadium chloroperoxidase.

Amino Acid Sequence↗
PubMed1994

The stability and steady-state kinetics of vanadium chloroperoxidase from the fungus Curvularia inaequalis.

In this article we report on the steady-state kinetics of the chlorination and the stability of the vanadium chloroperoxidase from the fungus Curvularia inaequalis. The data show that the kinetics of this enzyme resemble that of the vanadium bromoperoxidase from the seaweed Ascophyllum nodosum. At low pH, chloride inhibited the enzyme, but the inhibition was of a dual nature. At pH 4.1 a mixed type of inhibition by chloride with respect to hydrogen peroxide was observed whereas at pH 3.1 the nature of the inhibition became competitive. The log Km for hydrogen peroxide decreased linearly with pH with a slope of -1 in the pH range 3-5. A reaction mechanism is presented to explain the observed data. We also showed that this class of enzymes is inhibited by nitrate. At pH 5.5, nitrate inhibits the chlorination reaction competitively with respect to chloride (Ki = 2 mM) and uncompetitively with respect to hydrogen peroxide. Furthermore, we showed that the enzyme produces HOCl as a reaction product. The enzyme exhibited a high thermostability (tm of 90 degrees C) and displayed high stability in organic solvents (solutions of 40% methanol, ethanol or 2-propanol) and moderate stability in the presence of the chaotropic agent guanidine/HCl (G1/2, the concentration of guanidine/HCl at which the enzyme activity was half the original activity was 3.7 M).

Chloride Peroxidase↗
PubMed1994

Optical spectrum of myeloperoxidase. Origin of the red shift.

The optical spectrum of reduced myeloperoxidase (EC 1.11.1.7) displays an unusual red shift of the Soret band which is at 472 nm and the alpha-band which is at 636 nm. The spectral properties of myeloperoxidase can be modified by means of acid treatment. Upon short exposure to acid (pH 1.7) the red-shifted optical absorption spectrum of the reduced enzyme (lambda max at 472 nm) was blue-shifted (lambda max at 448 nm) but the spectrum of the reduced state could be restored by increasing the pH. By contrast, the resonance Raman spectra of both the oxidized and reduced enzyme are essentially the same at both pH 1.7 and pH 7.0. This shows that the optical spectrum and the resonance Raman spectrum are not directly correlated, which we interpret to indicate that the reversible effects of lower pH primarily affect the excited-state energy levels of the macrocycle. The EPR spectrum of the oxidized enzyme showed a reversible conversion from a high-spin rhombic spectrum (gx = 6.7, gy = 5.2) at neutral pH into a more axial high-spin spectrum (gx = gy = 5.8) at low pH. Upon prolonged exposure to acid (20 min) optical absorbance spectra, EPR spectra, resonance Raman spectra and the chlorinating activity were irreversibly affected. We propose that a negatively charged protonatable residue in the proximity of a pyrrole nucleus of the haem group is present that imposes the red shift in the optical absorption spectrum. This is consistent with the available X-ray structure data.

Electron Spin Resonance Spectroscopy↗
PubMed1994

Effects of cytochrome c on the oxidation of reduced cytochrome c oxidase by hydrogen peroxide.

The oxidation of the redox centres in reduced cytochrome c oxidase by hydrogen peroxide was studied by stopped-flow spectrophotometry in the absence and presence of reduced cytochrome c. The oxidation rate of cytochrome a decreased in the presence of cytochrome c. This effect was more pronounced at low than at high ionic strength. Cytochrome c did not influence the time-course of the oxidation of CuA or cytochrome a3. The oxidation of cytochrome c itself was faster at low ionic strength. The results suggest that the effect of cytochrome c is caused by re-reduction of cytochrome a by cytochrome c, the rate of which is dependent upon the ionic strength. We conclude that cytochrome a and cytochrome c are in equilibrium and that the equilibrium constant depends on the ionic strength. At low ionic strength, as a complex is formed between cytochrome c and cytochrome c oxidase, cytochrome a is more reduced than at high ionic strength conditions, when no such complex exists. Since CuA is oxidized at the same rate whether cytochrome c is present or not, we conclude that electron transfer from cytochrome a or cytochrome c to CuA is slower than electron transfer from CuA to cytochrome a or/and to the cytochrome a2-CuB couple.

Copper↗
PubMed1994

Resonance Raman microspectroscopic characterization of eosinophil peroxidase in human eosinophilic granulocytes.

A resonance Raman microspectroscopic study is presented of eosinophil peroxidase (EPO) in human eosinophilic granulocytes. Experiments were carried out at the single cell level with laser excitation in Soret-, Qv-, and charge transfer absorption bands of the active site heme of the enzyme. The Raman signal obtained from the cells was almost exclusively due to EPO. Methods were developed to determine depolarization ratios and excitation profiles of Raman bands of EPO in situ. A number of Raman band assignments based on earlier experiments with isolated EPO have been revised. The results show that in agreement with literature on isolated eosinophil peroxidase, the prosthetic group of the enzyme in the (unactivated) cells is a high spin, 6-coordinated, ferric protoporphyrin IX. The core size of the heme is about 2.04 A. The proximal and distal axial ligands are most likely a histidine with the strong imidazolate character typical for peroxidases, and a weakly bound water molecule, respectively. The data furthermore indicate that the central iron is displaced from the plane of the heme ring. The unusual low wavenumber Raman spectrum of EPO, strongly resembling that of lactoperoxidase, intestinal peroxidase and myeloperoxidase, suggests that these mammalian peroxidases are closely related, and characterized by, as yet unspecified, interactions between the peripheral substituents and the protein, different from those found in other protoheme proteins.

Binding Sites↗
PubMed1993

The vanadium chloroperoxidase from the fungus, Curvularia inaequalis. Evidence for the involvement of a histidine residue in the binding of vanadate.

The binding of vanadate to the novel vanadium chloroperoxidase from C. inaequalis was investigated. Reconstitution experiments of apo-chloroperoxidase by vanadate at different pH values showed that in the pH 6-7 range an acid/base group is present which affects the binding of the vanadate. It is proposed that this group is a histidine. This hypothesis was tested by specifically modifying this residue using diethylpyrocarbonate. In the apo-enzyme 9 histidines were modified, whereas in the holo-enzyme 6 histidines were modified. Modification with diethylpyrocarbonate had no effect on the chlorinating activity of the holo-enzyme, but when the apo-enzyme was modified the reactivation by vanadate was strongly inhibited. We conclude that histidine in the active site of chloroperoxidase is involved in the binding of vanadate.

Binding Sites↗
PubMed1993

Interaction of myeloperoxidase with peroxynitrite. A comparison with lactoperoxidase, horseradish peroxidase and catalase.

Polymorphonuclear neutrophils generate both nitric oxide and superoxide and these molecules can combine to form peroxynitrite. Neutrophils also contain myeloperoxidase which reacts with peroxynitrous acid (HOONO). On mixing myeloperoxidase with HOONO compound II was formed. Compound I could not be detected as an intermediate. The apparent second-order rate constant of formation of compound II was strongly pH-dependent (2.5 x 10(5) M-1 x s-1 at pH 8.9 and 6.2 x 10(6) M-1 x s-1 at pH 7.2). The pKa of this effect is 6.9 and it was concluded that the enzyme reacts with the protonated form of the peroxide, that is peroxynitrous acid, with a pH-independent second-order rate constant of 2.0 x 10(7) M-1 x s-1 at 12 degrees C. The interaction of HOONO with lactoperoxidase was studied for comparison. As was observed for myeloperoxidase, compound I could not be detected as an intermediate. The apparent second-order rate constant of compound II formation is pH-dependent and is 3.3 x 10(5) M-1 x s-1 at pH 7.4 and 8.4 x 10(4) M-1 x s-1 at pH 9.0. In contrast, horseradish peroxidase reacts with HOONO to form compound I, which is subsequently followed by the formation of compound II. The second-order rate constant for the formation of compound I is 3.2 x 10(6) M-1 x s-1 and is pH-dependent, the pKa for this effect is 6.8. Catalase (up to 3 microM) does not affect the rate of decomposition of peroxynitrite and no compound I formation is observed. Since nitrite may be present in the peroxynitrite preparation and to discriminate between the reaction of the enzyme with nitrite or peroxynitrite, the effect of nitrite on myeloperoxidase was studied. The dissociation constant for the myeloperoxidase-nitrite complex is pH-dependent and has values of 580 microM at pH 6.0 and 55 mM at pH 8.5.

Catalase↗
PubMed1993

The chloroperoxidase from the fungus Curvularia inaequalis; a novel vanadium enzyme.

The presence of vanadium-containing bromoperoxidases in various types of seaweed is well-documented. We now report that the terrestrial fungus Curvularia inaequalis excretes a novel chloroperoxidase which also contains vanadium as a prosthetic group. The chloroperoxidase is excreted in the medium as the only protein and is, therefore, almost purely obtained. Atomic absorption spectroscopy measurements showed that the chloroperoxidase contained vanadium, which was essential for enzymatic activity, in a stoichiometry of 1 mol vanadium per mol of enzyme. When the fungus was grown in media containing low concentrations of vanadate (VO4(3-)) or when vanadate was absent, the enzyme was excreted in an apoform. Addition of vanadate to the apoenzyme purified from the medium, dialyzed holo-enzyme or growth medium led to incorporation of the metal and to a subsequent increase in specific activity from 0.7 to about 7.5 units/mg. The reduced enzyme showed an axially symmetric EPR spectrum (g(o) = 1.971, Ao = 91.7 x 10(-4) cm-1) with 16 hyperfine lines that is essentially the same as the EPR spectrum of the vanadium-containing bromoperoxidase of the seaweed Ascophyllum nodosum. This demonstrates that the active sites in the two enzymes are very similar. The chlorinating and brominating activities of the chloroperoxidase from C. inaequalis were also studied and compared to those of the vanadium bromoperoxidase from A. nodosum. The chlorinating reaction catalyzed by the chloroperoxidase had a pH optimum around 5.5 and the Km for Cl- was small (0.25 mM at pH 4.5), but the logarithm of its value increased linearly with increasing pH. At high bromide concentrations, the pH optima of chloroperoxidase and bromoperoxidase in the brominating reaction were about the same (5.5). However, at low bromide concentrations the pH optimum of the chloroperoxidase was at higher pH values than that of the bromoperoxidase.

Chloride Peroxidase↗
PubMed1993

Lignin peroxidase L3 from Phlebia radiata. Pre-steady-state and steady-state studies with veratryl alcohol and a non-phenolic lignin model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol.

The catalytic cycle of lignin peroxidase (LiP, ligninase) isozyme L3 from the white-rot fungus Phlebia radiata was investigated using stopped-flow techniques. Veratryl (3,4-dimethoxybenzyl) alcohol and a lignin model compound, non-phenolic beta-O-4 dimer 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol, were used as electron donors. This is the first report on the detailed kinetic analysis of a LiP-catalysed C alpha-C beta bond cleavage of the dimer, representing the major depolymerisation reaction in the lignin polymer. The native enzyme showed a typical heme peroxidase absorbance spectrum with a Soret maximum at 407 nm. Following the reaction with H2O2, the Soret band decreased in absorbance, shifted to 403 nm and then to 421 nm, demonstrating the formation of compound I followed by the formation of compound II, respectively. Similar results have been reported for the LiP from Phanerochaete chrysosporium upon reaction with H2O2. However, compound I of L3 was more stable in the absence of additional electron donors. The second-order rate constant of compound I formation by H2O2 was determined to be 6 x 10(5) M-1 s-1 and was the same at pH 3.0 and 6.0. Compound I was rapidly reduced to compound II and further to native enzyme when either veratryl alcohol or the beta-O-4 dimer was supplied as electron donor and in both cases veratraldehyde appeared as the major product. At pH 6.0, the second-order rate constant for compound II formation was similar with either veratryl alcohol or the beta-O-4 dimer (6.7 x 10(3) and 6.5 x 10(3) M-1 s-1, respectively). At pH 3.0 formation of compound II with either reductant proceeded so rapidly that determination of the respective rate constants was not possible. The results point to identical catalytic cycles of L3 with veratryl alcohol or the beta-O-4 dimer involving both compounds I and II as intermediates and participation of the same veratryl alcohol radical as the most appropriate reductant for compound II. Chemical evidence of such a radical, formed after the initial LiP-catalysed one-electron oxidation of beta-O-4 dimeric lignin models, is presented in a separate article [Lundell, T., Schoemaker, H., Hatakka, A. & Brunow, G. (1993) Holzforschung, in the press]. The catalytic redox-cycle and the oxidation mechanism presented here reconcile seemingly contradictory results obtained in previous studies on LiP kinetics during the last decade.

Benzyl Alcohols↗