Fructose induces and glucose represses chloroperoxidase mRNA levels

The fungus Caldariomyces fumago can be induced to secrete the heme protein chloroperoxidase at levels of 500 mg/liter. Chloroperoxidase synthesis is controlled at the mRNA level. Glucose strongly represses production of chloroperoxidase mRNA and protein, whereas fructose induces both to high levels. Chloroperoxidase has been partially sequenced by automated Edman degradation of tryptic peptides. Based on this amino acid sequence data, a 2-fold degenerate, 29-base oligonucleotide (29-mer) complementary to chloroperoxidase mRNA was synthesized. Polyadenylated RNA, purified from C. fumago, was used as substrate for cDNA synthesis using the 29-mer as primer. cDNAs were made double-stranded and cloned into plasmid pBR322 by conventional methods. Screening the resultant cDNA bank by colony hybridization with the 29-mer as probe showed that 18% of the clones contained the 29-mer sequence. Dideoxy sequencing of one clone (pMA340) identified it as part of the coding region for chloroperoxidase by comparison with known amino acid sequences. In addition, the amino-terminal coding region of clone pMA340 reveals a putative signal peptide for chloroperoxidase. Clone pMA340 was then used in Northern analysis of chloroperoxidase mRNA levels under conditions which induce and repress enzyme secretion.     

Chloroperoxidase-catalyzed halogenation of trans-cinnamic acid and its derivatives

Several 2,3-unsaturated carboxylic acids, such as trans-cinnamic acid and its derivatives, were found to be halogenated by chloroperoxidase of Caldariomyces fumago in the presence of hydrogen peroxide and either Cl- or Br-. Cinnamic acid, 4-hydroxycinnamic acid, 4-methoxycinnamic acid, and 3,4-dimethoxycinnamic acid were suitable substrates of chloroperoxidase, and were converted to 2-halo-3-hydroxycarboxylic acid, 2,3-dihydroxycarboxylic acid, decarboxylated halohydrin, or decarboxylated halocompound. However, 4-nitrocinnamic acid and 4-chlorocinnamic acid having electron-attracting groups did not serve as a substrate of the enzyme. The enzyme also did not act on acrylic acid, acrylamide, crotonic acid, fumaric acid, etc. From these data, the enzymatic reactions of chloroperoxidase, concerning the substrate specificity, stereoselectivity, and the reaction mechanism, are discussed on the basis of current knowledge regarding the reaction mechanism of the enzyme. Also they are compared with the chemical reactions of molecular halogen and hypohalous acid.     

Kinetics of the oxidation of ascorbic acid, ferrocyanide and p-phenolsulfonic acid by chloroperoxidase compounds I and II

For the first time elementary reactions involving chloroperoxidase compounds I and II have been investigated. A multi-mixing stopped-flow apparatus was used to study the kinetics of the reactions of compounds I and II with ascorbic acid, ferrocyanide and p-phenolsulfonic acid. The second-order rate constants of the reactions of both compounds with all three substrates were determined between pH 3 and pH 7. In all cases the rate constants decrease with increasing pH. The reactions of p-phenolsulfonic acid are influenced by a catalytically important group on both compounds I and II with a pKa of 3.7 +/- 0.2. With ascorbic acid and ferrocyanide as substrates, a decrease in rate was observed upon ionization of the substrate. Comparisons with horseradish peroxidase show that chloroperoxidase is a much less efficient peroxidatic enzyme. The kinetic data were used to calculate the percentage composition of the mixture of chloroperoxidase species which contribute to the spectra measured during the turnover with ascorbate as substrate.     

Identification of the fifth axial heme ligand of chloroperoxidase

Chloroperoxidase from Caldariomyces fumago catalyzes the peroxidative chlorination of organic acceptor molecules. From a variety of spectroscopic data, it had long been thought that chloroperoxidase possessed an active site structure similar to that of cytochrome P-450cam. Resonance Raman studies conducted with isotopically substituted enzyme proved conclusively that the fifth axial ligand to the ferriprotoporphyrin IX moiety of chloroperoxidase is indeed a cysteine thiolate (Bangcharoenpaurpong, O., Champion, P. M., Hall, K. S., and Hager, L. P. (1986) Biochemistry 25, 2374-2378). In this study, Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was used to ascertain which of the 3 cysteine residues in the primary structure of chloroperoxidase serves as the fifth axial heme ligand; two of the cysteine residues were earlier shown to be involved in a disulfide linkage. Apoprotein was labeled under denaturing conditions with 5,5'-dithiobis(2-nitrobenzoic acid). A unique peptide, containing the thionitrobenzoate adduct, was isolated via reverse phase HPLC following digestion with endoproteinase Glu-C. Amino acid and Edman sequence analysis revealed the fifth axial ligand in chloroperoxidase to be cysteine 29. Under reducing and denaturing conditions, incubation of apochloroperoxidase with Ellman's reagent resulted in 3 labeled residues. Proteolysis and isolation of the labeled peptides using reverse phase HPLC and subsequent Edman sequence analysis detected and identified the thionitrobenzoate adducts of each of the three cysteinyl peptides of chloroperoxidase.     

Cloning and high-level expression of a chloroperoxidase gene from Pseudomonas pyrrocinia in Escherichia coli

A chloroperoxidase gene from Pseudomonas pyrrocinia was cloned into Escherichia coli using the cosmid vector pJB8. The gene coding for the chloroperoxidase could be localized to a 1.5 kb fragment of DNA which was subcloned into the high-copy-number plasmid pUC18. In one subclone increased halogenating activity could be found which was 570-fold greater than in P. pyrrocinia. The halogenating enzyme was identified as the chloroperoxidase by SDS-polyacrylamide gel electrophoresis.     

Chloroperoxidase from Streptomyces lividans: isolation and characterization of the enzyme and the corresponding gene.

For the first time, a halogenating enzyme which is not known to produce halogenated metabolites has been isolated from a bacterial strain. The gene encoding the nonheme chloroperoxidase (CPO-L) from Streptomyces lividans TK64 was cloned, and its gene product was characterized. S. lividans TK64 produced only very small amounts of the enzyme. After cloning of the gene into Streptomyces aureofaciens Tü24-88, the enzyme was overexpressed up to 3,000-fold. Based on the overexpression, a simple purification procedure using acid precipitation and hydrophobic interaction chromatography was developed. Thus, 54 mg of homogeneous CPO-L could be obtained from 27 g (wet weight) of mycelium. The native enzyme has a molecular weight of 64,000 and consists of two identical subunits. The enzyme does not exhibit an absorption peak in the Soret region of the optical spectrum. X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metal ions in equimolar amounts. CPO-L showed cross-reaction with antibodies raised against the nonheme chloroperoxidase from Pseudomonas pyrrocinia but not with antibodies raised against CPO-T from S. aureofaciens Tü24. CPO-L exhibits substrate specificity only for chlorination, not for bromination. Therefore, monochlorodimedone is only brominated by CPO-L, whereas indole is brominated and chlorinated. The functional chloroperoxidase gene was located on a 1.9-kb SalI DNA fragment. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide of 276 amino acids. The overall identity of the amino acid sequence to that of chloroperoxidase from P. pyrrocinia was 71%, whereas that to bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 was only 42%.     

On the mechanism of chlorination by chloroperoxidase

Spectral-scan results obtained on the millisecond time scale are reported for reactions of chloroperoxidase with peracetic acid and chloride ion in both the presence and the absence of monochlorodimedone. A multimixing experiment is performed in which stoichiometric amounts of chloroperoxidase and peracetic acid are premixed for 0.7 s before the resultant compound I is reacted with chloride ion. The combined results show that the only detectable enzyme intermediate species is compound I (except in very late stages of the reaction), that the disappearance of compound I is accelerated by the presence of chloride ion, and that it is further accelerated if both chloride and monochlorodimedone are present. It is concluded that compound I is an obligate intermediate species in the reaction. Experiments are performed on the reaction of monochlorodimedone with hypochlorous acid in both the presence and the absence of added chloride ion, but in the absence of chloroperoxidase. The presence of chloride ion greatly accelerates the reaction rate apparently by setting off a chlorine chain reaction. This reaction would be important in the enzyme-catalyzed reaction if hypochlorous acid were liberated into the solution. A careful analysis of steady-state kinetic results shows that in the chlorination of monochlorodimedone at least, liberation of free hypochlorous acid is not important in the enzyme-catalyzed pathway. Rather the reaction proceeds from compound I to formation of iron(III)-OCl by chloride ion addition to the ferryl oxygen atom. This obligate intermediate species then chlorinates the substrate. It is well described as enzyme-activated hypochlorous acid, in which replacement of the proton in HOCl by the heme iron ion produces a Cl+ species of great potency. Thus the enzyme controls chlorination of monochlorodimedone rather than unleashing an uncontrolled chain reaction in which it would be rapidly destroyed.     

A colorimetric assay for immobilized chloroperoxidase

A rapid and sensitive colorimetric assay was developed for the estimation of chloroperoxidase activity. N,N,N',N'-Tetramethyl-p-phenylenediamine was chosen from four potential chromogenic substrates because the blue product resulting from chloroperoxidase conversion gave the highest molar absorption. This product exhibited two absorbance maxima, at 563 and 610 nm. Activity was monitored at 563 nm, and the product absorbance was stable for at least 1 h at 10 degrees C after treatment with an equal volume of a mixture (40:1) of methanol and phosphoric acid (85% w/v), pH 2. The linear range of the assay with respect to enzyme amount was determined. The assay was developed using soluble chloroperoxidase but worked well with the enzyme immobilized on glass beads.     

Oxidation-reduction potential measurements on chloroperoxidase and its complexes.

The oxidation-reduction potential of chloroperoxidase, an enzyme which catalyzes peroxidative chlorination, bromination, and iodination reactions, has been investigated. In addition to catalyzing biological halogenation reactions, chloroperoxidase is unusual in that the carbon monoxide complex of ferrous chloroperoxidase shows the typical long wavelength Soret absorption associated with P-450 hemoproteins. The pH dependence of the chloroperoxidase oxidation-reduction potential shows a discontinuity around pH 4.7. Similarly, measurements of the affinity of ferrous chloroperoxidase for carbon monoxide monitored both by spectroscopic and potentiometric titration exhibit a discontinuity in the pH 4.7 region. Oxidation-reduction potential measurements on chloroperoxidase in a CO atmosphere also show a discontinuous pH profile. These results suggest that ferrous chloroperoxidase undergoes reversible modification at low pH and that these changes are reflected in the oxidation-reduction potential. The oxidation-reduction potential of chloroperoxidase at pH 6.9 is - 140 mV, close to that measured for cytochrome P-450cam in the presence of substrate. The oxidation-reduction potential of chloroperoxidase at pH 2.7, the pH optimum for enzymatic chlorination, is +150 mV. The oxidation-reduction potentials of the halide complexes of chloroperoxidase (chloride, bromide, and iodide) are essentially identical with the potential measurements on the native enzyme. These observations suggest that, although halide anions bind to the enzyme, they probably do not bind as an axial ligand to the heme ferric iron.     

Examining the role of glutamic acid 183 in chloroperoxidase catalysis

Site-directed mutagenesis has been used to investigate the role of glutamic acid 183 in chloroperoxidase catalysis. Based on the x-ray crystallographic structure of chloroperoxidase, Glu-183 is postulated to function on distal side of the heme prosthetic group as an acid-base catalyst in facilitating the reaction between the peroxidase and hydrogen peroxide with the formation of Compound I. In contrast, the other members of the heme peroxidase family use a histidine residue in this role. Plasmids have now been constructed in which the codon for Glu-183 is replaced with a histidine codon. The mutant recombinant gene has been expressed in Aspergillus niger. An analysis of the produced mutant gene shows that the substitution of Glu-183 with a His residue is detrimental to the chlorination and dismutation activity of chloroperoxidase. The activity is reduced by 85 and 50% of wild type activity, respectively. However, quite unexpectedly, the epoxidation activity of the mutant enzyme is significantly enhanced approximately 2.5-fold. These results show that Glu-183 is important but not essential for the chlorination activity of chloroperoxidase. It is possible that the increased epoxidation of the mutant enzyme is based on an increase in the hydrophobicity of the active site.     

Proton nuclear Overhauser effect study of the heme active site structure of chloroperoxidase.

Chloroperoxidase, a glycoprotein from the mold Caldariomyces fumago, has been investigated in its ferric low-spin cyanide-ligated form through use of nuclear Overhauser effect (NOE) spectroscopy to provide information on the heme pocket electronic/molecular structure. Spin-lattice relaxation times for the hyperfine-shifted heme resonances were found to be three times less than those in horseradish peroxidase. This must reflect a slower electronic relaxation rate for chloroperoxidase than for horseradish peroxidase as a consequence of axial ligation of cysteine in the former versus histidine in the latter enzyme. Isoenzymes A1 and A2 of chloroperoxidase show the largest chemical shift differences near the heme propionate on the basis of NOE measurements. This suggests that the primary structure differences for the two isoenzymes are communicated to the heme group through the ring propionate substituents. A downfield peak has been detected in chloroperoxidase with chemical shift, T1, and line width characteristics similar to those of the C epsilon-H proton of the distal histidine residue. The NOE pattern and T1's of the peaks in the 0.0 to -5.0 ppm upfield region are consistent with the presence of an arginine amino acid residue in the heme pocket near either the 1-CH3 or 3-CH3 group. Existence of catalytically important distal histidine and arginine amino acid residues in chloroperoxidase shows it to be structurally similar to peroxidases rather than to the often compared monooxygenase, cytochrome P-450. This result supports the earlier conclusions of Sono et al. [Sono, M., Dawson, J.H., Hall, K., & Hager, L.P. (1986) Biochemistry 25, 347-356].     

Evidence for a radical mechanism of halogenation of monochlorodimedone catalyzed by chloroperoxidase

A radical species of monochlorodimedone has been characterized by its high reactivity with molecular O2. Horseradish peroxidase greatly accelerated O2 uptake by acidic solutions of this substrate; the enzymatic reaction required exogenous H2O2 only with freshly prepared substrate solutions, and the total substrate oxidized was equal to the sum of H2O2 added and O2 consumed. However, with excess Br- and horseradish peroxidase, or high Br- or Cl- and chloroperoxidase, a 1:1 stoichiometry between H2O2 and substrate was observed. In the absence of halide, the stoichiometry of the chloroperoxidase-catalyzed oxidation of monochlorodimedone changed to two molecules of the organic donor per H2O2. Moreover, in the absence of halide, at substrate:H2O2 ratios greater than 2.0, chloroperoxidase catalyzed significant O2 uptake; this enzyme-dependent autoxidation of monochlorodimedone also occurred in the presence of Cl- or Br-, when H2O2 was limiting. These data, and recent evidence from this laboratory for free hypohalous acid as the first product of chloroperoxidase-catalyzed halide oxidation [B. W. Griffin (1983) Biochem. Biophys. Res. Commun. 116, 873-879], strongly support a mixed enzymatic/nonenzymatic radical chain process as the mechanism for halogenation of monochlorodimedone by chloroperoxidase. Both horseradish peroxidase and chloroperoxidase can catalyze either bromination or oxidation of this substrate, depending on the experimental conditions. Implications of these results for the mechanism of HOCl formation catalyzed by chloroperoxidase are considered.     

Mechanism of azide binding to chloroperoxidase and horseradish peroxidase: use of an iodine laser temperature-jump apparatus

The kinetics of azide binding to chloroperoxidase have been studied at eight pH values ranging from 3.0 to 6.6 at 9.5 +/- 0.2 degrees C and ionic strength of 0.4 M in H2O. The same reaction was studied in D2O at pD 4.36. In addition, results were obtained on azide binding to horseradish peroxidase at pD 4.36 and pH 4.56. Typical relaxation times were in the range 10-40 microseconds. The value of kH/kD(on) for chloroperoxidase is 1.16, and kH/kD(off) is 1.7; corresponding values for horseradish peroxidase are 1.10 and 2.4. The H/D solvent isotope effects indicate proton transfer is partially rate controlling and is more important in the dissociation of azide from the enzyme-ligand complex. A mechanism is proposed in which hydrazoic acid binds to chloroperoxidase in a concerted process in which its proton is transferred to a distal basic group. Hydrogen bonding from the newly formed distal acid to the bound azide facilitates formation of hydrazoic acid as the leaving group in the dissociation process. The binding rate constant data, kon, can be fit to the equation kon = k3/(1 + KA/[H+]), where k3 = 7.6 X 10(7) M-1 S-1 and KA, the dissociation constant of hydrazoic acid, is 2.5 X 10(-5) M. The same mechanism probably is valid for the ligand binding to horseradish peroxidase.     

Chloroperoxidase-catalyzed enantioselective oxidation of methyl phenyl sulfide with dihydroxyfumaric acid/oxygen or ascorbic acid/oxygen as oxidants

The chloroperoxidase catalyzed oxidation of methyl phenyl sulfide to (R)-methyl phenyl sulfoxide was investigated, both in batch and membrane reactors, using as oxidant H2O2, or O2 in the presence of either dihydroxyfumaric acid or ascorbic acid. The effects of pH and nature and concentration of the oxidants on the selectivity, stability, and productivity of the enzyme were evaluated. The highest selectivity was displayed by ascorbic acid/O2, even though the activity of chloroperoxidase with this system was lower than that obtained with the others. When the reaction was carried out in a membrane reactor, it was possible to reuse the enzyme for several conversion cycles. The results obtained with ascorbic acid/O2 and dihydroxyfumaric acid/O2 as oxidants do not seem to be compatible with either a mechanism involving hydroxyl radicals as the active species or with the hypothesis that oxidation occurs through the initial formation of H2O2. Copyright 1999 John Wiley & Sons, Inc.     

Mechanisms of 1,3-butadiene oxidations to butadiene monoxide and crotonaldehyde by mouse liver microsomes and chloroperoxidase

NADPH-dependent oxidation of 1,3-butadiene by mouse liver microsomes or H2O2-dependent oxidation by chloroperoxidase produced both butadiene monoxide and crotonaldehyde; methyl vinyl ketone and 2,3- and 2,5- dihydrofuran were not detected. The crotonaldehyde to butadiene monoxide ratio remained constant over time in both the microsomal and the chloroperoxidase reactions; however, much more crotonaldehyde was produced by chloroperoxidase than microsomes; crotonaldehyde was not detected when reference samples of butadiene monoxide were used in control incubations containing NADPH and microsomes or H2O2 and chloroperoxidase. Moreover, incubations of 1,3-butadiene with horseradish peroxidase and H2O2, or microsomes and H2O2 or arachidonic acid did not result in the oxidation of 1,3-butadiene. In microsomes, metabolite formation was dependent on incubation time, NADPH, and protein concentrations and did not change when the 1,3-butadiene pressure was varied between 24 and 52 cm Hg. Inclusion of the cytochrome P450 inhibitor 1-benzylimidazole inhibited 1,3-butadiene metabolism, but inclusion of KCN, catalase, or superoxide dismutase had no effect. These results support the role of cytochrome P450 in 1,3-butadiene oxidation by mouse liver microsomes. The formation of crotonaldehyde but not methyl vinyl ketone by cytochrome P450 or chloroperoxidase indicates regioselectivity in the oxygen transfer from the hemoproteins to 1,3-butadiene. The intermediates formed may undergo either ring closure to form butadiene monoxide or a hydrogen shift to form 3-butenal which tautomerizes to produce crotonaldehyde. Evidence for this tautomerization was obtained by the finding that 3-buten-1-ol, an alternative precursor of 3-butenal, was oxidized to crotonaldehyde under incubation conditions similar to that used for 1,3-butadiene.     

Oxygen binding to dithionite-reduced chloroperoxidase

Both the kinetics of ferric chloroperoxidase reduction by dithionite and the binding of molecular oxygen to ferrous chloroperoxidase have been studied. The oxyferrous chloroperoxidase decays spontaneously to the ferric enzyme. In addition the corresponding rapid-scan spectra have been recorded. The reduction reaction is caused by SO-.2 with a rate constant of (7.7 +/- 1.0) X 10(4) M-1 S-1. Oxygen binding occurs with a rate constant of (5.5 +/- 1.0) X 10(5) M-1 S-1 over the pH range 3.5-6. Oxyferrous chloroperoxidase has a Soret absorption peak at 428 nm and two partially resolved peaks at 555 nm and 588 nm. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous chloroperoxidase at 419, 545, 555 and 580 nm; between oxyferrous and ferric chloroperoxidase at 419, 487, 540, 609 and 682 nm.     

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.     

Amino acid metabolism in maize earshoots. Implications for assimilate preconditioning and nitrogen signaling

Nitrogen (N) is an essential requirement for kernel growth in maize (Zea mays); however, little is known about how N assimilates are metabolized in young earshoots during seed development. The objective of this study was to assess amino acid metabolism in cob and spikelet tissues during the critical 2 weeks following silking. Two maize hybrids were grown in the field for 2 years at two levels of supplemental N fertilizer (0 and 168 kg N/ha). The effects of the reproductive sink on cob N metabolism were examined by comparing pollinated to unpollinated earshoots. Earshoots were sampled at 2, 8, 14, and 18 d after silking; dissected into cob, spikelet, and/or pedicel and kernel fractions; then analyzed for amino acid profiles and key enzyme activities associated with amino acid metabolism. Major amino acids in the cob were glutamine (Gln), aspartic acid (Asp), asparagine (Asn), glutamate, and alanine. Gln concentrations dropped dramatically from 2 to 14 d after silking in both pollinated and unpollinated cobs, whereas all other measured amino acids accumulated over time in unpollinated spikelets and cobs, especially Asn. N supply had a variable effect on individual amino acid levels in young cobs and spikelets, with Asn being the most notably enhanced. We found that the cob performs significant enzymatic interconversions among Gln, alanine, Asp, and Asn during early reproductive development, which may precondition the N assimilate supply for sustained kernel growth. The measured amino acid profiles and enzymatic activities suggest that the Asn to Gln ratio in cobs may be part of a signal transduction pathway involving aspartate aminotransferase, Gln synthetase, and Asn synthetase to indicate plant N status for kernel development.     

Effects of chloroperoxidase and hydrogen peroxide on the viabilities of Aspergillus flavus conidiospores

The effects of chloroperoxidase [EC 1.1.1.10] and hydrogen peroxide on the viabilities of quiescent and germinating conidiospores of an aflatoxigenic fungus, Aspergillus flavus, were determined. Hydrogen peroxide was found moderately lethal and chloroperoxidase produced a 30-fold increase in the lethality of hydrogen peroxide to germinating conidia, which were 75-fold more susceptible to chloroperoxidase than were quiescent conidia. According to infrared examinations of fungal corpses, mortality occurred by oxidation rather than peroxidative chlorination.