Purification and characterization of a novel bacterial non-heme chloroperoxidase from Pseudomonas pyrrocinia

The first bacterial chloroperoxidase that is capable of catalyzing the chlorination of indole to 7-chloroindole was detected in Pseudomonas pyrrocinia ATCC 15958, a bacterium that produces the antifungal antibiotic pyrrolnitrin (Wiesner, W., van Pée, K.H., and Lingens, F. (1986) FEBS Lett. 209, 321-324). Here we describe the purification and characterization of this bacterial non-heme chloroperoxidase. The enzyme was purified by DEAE-cellulose chromatography at different pH values, molecular sieve chromatography, and Bio-Gel HTP hydroxylapatite. After the last purification step, chloroperoxidase was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. Based on gel filtration and ultracentrifugation results, the molecular weight of the enzyme was 64,000 +/- 3,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band with the mobility of a 32,000 molecular weight species. Therefore, in solution at neutral pH, this chloroperoxidase is a dimer. The enzyme did not exhibit any absorbance in the visible region of the spectrum. The isoelectric point was 4.1. Chloroperoxidase was specific for I-, Br-, and Cl- and was not inhibited by azide, but was inhibited by cyanide and F-. This procaryotic chloroperoxidase catalyzed the bromination of monochlorodimedone but not its chlorination and has no peroxidase or catalase activity. The pH optimum of the enzyme was between 4.0 and 4.5, and the enzyme was stable between pH 3.5 and 8.5 and showed no loss of activity when incubated at 60 degrees C for 2 h. Chloroperoxidase also chlorinated 4-(2-amino-3-chlorophenyl) pyrrole to yield aminopyrrolnitrin, the immediate precursor of pyrrolnitrin. This suggests very strongly that chloroperoxidase is involved in the biosynthesis of the antibiotic pyrrolnitrin.     

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.     

Purification of bromoperoxidase from Pseudomonas aureofaciens.

A Bromoperoxidase has been isolated and purified from Pseudomonas aureofaciens ATCC 15926 mutant strain ACN. The purified enzyme was homogeneous as determined by polyacrylamide gel electrophoresis and ultracentrifugation. This bromoperoxidase can utilize bromide ions in the presence of hydrogen peroxide and a halogen acceptor for the catalytic formation of carbon-halogen bonds. The homogeneous enzyme also has peroxidase and catalase activity. Based on the results from gel filtration and ultracentrifugation, the molecular weight of this procaryotic bromoperoxidase is 155,000 to 158,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band having the mobility of a 77,000-molecular-weight species. We thus conclude that this bromoperoxidase exists in solution as a dimeric species. The heme prosthetic group of bromoperoxidase is ferriprotoporphyrin IX. The spectral properties of the native and reduced enzyme are reported. This bromoperoxidase is the first halogenating enzyme purified from procaryotic sources.     

Peroxide oxidation of indole to oxindole by chloroperoxidase catalysis.

In the presence of chloroperoxidase, indole was oxidized by H2O2 to give oxindole as the major product. Under most conditions oxindole was the only product formed, and under optimal conditions the conversion was quantitative. This reaction displayed maximal activity at pH 4.6, although appreciable activity was observed throughout the entire pH range investigated, namely pH 2.5-6.0. Enzyme saturation by indole could not be demonstrated, up to the limit of indole solubility in the buffer. The oxidation kinetics were first-order with respect to indole up to 8 mM, which was the highest concentration of indole that could be investigated. On the other hand, 2-methylindole was not affected by H2O2 and chloroperoxidase, but was a strong inhibitor of indole oxidation. The isomer 1-methylindole was a poor substrate for chloroperoxidase oxidation, and a weak inhibitor of indole oxidation. These results suggest the possibility that chloroperoxidase oxidation of the carbon atom adjacent to the nitrogen atom in part results from hydrogen-bonding of the substrate N-H group to the enzyme active site.     

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%.     

Purification and properties of bromoperoxidase from Pseudomonas pyrrocinia

A bromoperoxidase was purified and partially characterized from Pseudomonas pyrrocinia ATCC 15958, a bacterium that produces the antifungal antibiotic pyrrolnitrin. The purified enzyme preparation was homogeneous as determined by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular mass of the enzyme was estimated to be 154 kDa +/- 3 kDa as determined by gel filtration and ultracentrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band with the mobility of a 76-kDa species. Therefore, in solution at neutral pH, bromoperoxidase exists as a dimeric species. The isoelectric point was 5.0. The prosthetic group of this procaryotic bromoperoxidase was ferriprotoporphyrin IX. The spectral properties of the native and reduced enzyme are reported. The purified enzyme showed brominating as well as peroxidase and catalase activity.     

Inhibition of vanadium chloroperoxidase from the fungus Curvularia inaequalis by hydroxylamine, hydrazine and azide and inactivation by phosphate

The first detailed inhibition study of recombinant vanadium chloroperoxidase (rVCPO) using hydroxylamine, hydrazine and azide has been carried out. Hydroxylamine inhibits rVCPO both competitively and uncompetitively. The competitive inhibition constant K(ic) and the uncompetitive inhibition constant K(iu) see are 40 and 80 microM, respectively. The kinetic data suggest that rVCPO may form a hydroxylamido complex, hydroxylamine also seems to react with the peroxovanadate complex during turnover. The kinetic data show that the type of inhibition for hydrazine and azide is uncompetitive with the uncompetitive inhibition constant K(iu) of 350 microM and 50 nM, respectively, showing that in particular azide is a very potent inhibitor of this enzyme. Substitution of vanadate in the active site by phosphate also leads to inactivation of vanadium chloroperoxidase. However, the presence of H(2)O(2) clearly prevents the inactivation of the enzyme by phosphate. This shows that pervanadate is bound much more strongly to the enzyme than vanadate.     

Spectrum of chloroperoxidase compound I

When compound I of chloroperoxidase is formed from the native enzyme the absorption peak in the Soret region diminishes in intensity, and shifts to a maximum absorbance at 367 nm. This unusual Soret spectrum decreases in intensity in a linear fashion as the wavelength increases. The first visible spectrum of chloroperoxidase compound I is reported which has a peak at 689 nm as its most prominent feature.     

Chemical modification of chloroperoxidase with diethylpyrocarbonate. Evidence for the presence of an essential histidine residue

Chloroperoxidase from Caldariomyces fumago is well documented as an extremely versatile catalyst, and studies are currently being conducted to delineate the fine structural features that allow the enzyme to possess chemical and physical similarities to the peroxidases, catalases, and P-450 cytochromes. Earlier investigations of ligand binding to the heme iron of chloroperoxidase, along with the presence of an invariant distal histidine residue in the active site of peroxidases and catalases, have led to the hypothesis that chloroperoxidase also possesses an essential histidine residue that may participate in catalysis. To address this in a more direct fashion, chemical modification studies were initiated with diethylpyrocarbonate. Incubation of chloroperoxidase with this reagent resulted in a time-dependent inactivation of enzyme. Kinetic analysis revealed that the inactivation was due to a simple bimolecular reaction. The rate of inactivation exhibited a pH dependence, indicating that modification of a titratable residue with a pKa value of 6.91 was responsible for inactivation; this data provided strong evidence for histidine derivatization by diethylpyrocarbonate. To further support these results, inactivation due to cysteine, tyrosine, or lysine modification was ruled out. The stoichiometry of histidine modification was estimated by the increase in absorption at 246 nm, and it was found that more than 1 histidine residue was derivatized when chloroperoxidase was inactivated with diethylpyrocarbonate. However, it was shown that the rates of modification and inactivation were not equivalent. This was interpreted to reflect that both essential and nonessential histidine residues were modified by diethylpyrocarbonate. Kinetic analysis indicated that modification of a single essential histidine residue was responsible for inactivation of the enzyme. Studies with [14C]diethylpyrocarbonate provided stoichiometric support that derivatization of a single histidine inactivated chloroperoxidase. Based on sequence homology with cytochrome c peroxidase, histidine 38 was identified as a likely candidate for the distal residue. Molecular modeling, based on secondary structure predictions, allows for the construction of an active site peptide, and implicates a number of other residues that may participate in catalysis.     

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.     

Enzymatic bromination of the thiazole ring

Bromination of the thiazole ring by the enzyme chloroperoxidase, produced by the fungus Caldariomyces fumago, was demonstrated. Both 2-acetoacetamido-4-methylthiazole and 2-acetamidothiazole were brominated on C-5 of the thiazole ring in the presence of chloroperoxidase, bromide, and hydrogen peroxide in 0.06 M Phosphate solution at pH 3.0. No reaction occurred in the absence of enzyme.     

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.     

Cross-linked crystals of chloroperoxidase

Chloroperoxidase from Caldariomyces fumago was crystallized. The crystals were modified with several cross-linkers, but only glurataldehyde was able to produce catalytically active and insoluble crystals. Unlike other immobilized chloroperoxidase preparations, these catalytic crystals are more thermostable than the unmodified soluble enzyme. The enhanced stability is probably due to the structure conservation in the crystalline matrix. In addition, non-cross-linked chloroperoxidase crystals retained more activity than the soluble enzyme after incubation in an organic solvent with low water content. Although the cross-linked crystals were catalytically active, they showed lower specific activity than the soluble enzyme. This low activity may be due to non-specific reactions between the cross-linker and essential residues for catalysis. Alternative cross-linking strategies are discussed.     

Preparation and properties of ferrous chloroperoxidase complexes with dioxygen, nitric oxide, and an alkyl isocyanide. Spectroscopic dissimilarities between the oxygenated forms of chloroperoxidase and cytochrome P-450

Extensive spectroscopic investigations of chloroperoxidase and cytochrome P-450 have consistently revealed close similarities between these two functionally distinct enzymes. Although the CO-bound ferrous states were the first to display such resemblance, additional comparisons have focused on the native ferric and ferrous and the ligand-bound ferric derivatives of the enzymes. In order to test the extent to which the spectral properties of the two enzymes match each other, we have prepared the NO, alkyl isocyanide, and O2 adducts of ferrous chloroperoxidase, the latter two for the first time. As expected, the NO adducts of the two proteins have similar UV-visible absorption and magnetic circular dichroism spectra; the same behavior is observed for the alkyl isocyanide complexes. Unexpectedly, the dioxygen adduct of ferrous chloroperoxidase (i.e. Compound III), generated in cryogenic solvents at -30 degrees C by bubbling with O2, is spectrally distinct from oxy-P-450-CAM. Identification of this derivative as oxygenated chloroperoxidase is based on the following criteria: It is EPR-silent at 77 K. The bound O2 is dissociable as judged by the uniform conversion to the CO-bound form. Oxy-chloroperoxidase autoxidizes to form the native ferric enzyme without detectable intermediates at a rate comparable to that determined for oxy-P-450-CAM. Oxy-chloroperoxidase exhibits optical absorption (lambda nm (epsilon mM) = 354 (41), 430 (94), 554 (16.5), 587 (12.5)) and magnetic circular dichroism spectra that are clearly distinct from those of histidine-ligated heme proteins such as oxy-myoglobin or oxy-horseradish peroxidase. Surprisingly, several of its spectral properties, namely the red-shifted Soret peak and discrete alpha peak, are also unlike those of oxy-P-450-CAM. Since considerable evidence has accumulated supporting the ligation of an endogenous thiolate to the heme iron of chloroperoxidase, as has been established for the P-450 enzyme, the observed dissimilarities suggest that the electronic properties of the two dioxygen adducts are quite sensitive to differences in their active site heme environment. This, in turn may be related to the functional differences between the two enzymes.     

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.     

Identification of a novel phosphatase sequence motif.

We have identified a novel, conserved phosphatase sequence motif, KXXXXXXRP-(X12-54)-PSGH-(X31-54)-SRXXXXX HXXXD, that is shared among several lipid phosphatases, the mammalian glucose-6-phosphatases, and a collection of bacterial nonspecific acid phosphatases. This sequence was also found in the vanadium-containing chloroperoxidase of Curvularia inaequalis. Several lines of evidence support this phosphatase motif identification. Crystal structure data on chloroperoxidase revealed that all three domains are in close proximity and several of the conserved residues are involved in the binding of the cofactor, vanadate, a compound structurally similar to phosphate. Structure-function analysis of the human glucose-6-phosphatase has shown that two of the conserved residues (the first domain arginine and the central domain histidine) are essential for enzyme activity. This conserved sequence motif was used to identify nine additional putative phosphatases from sequence databases, one of which has been determined to be a lipid phosphatase in yeast.     

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.     

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.     

A simple, rapid, high yield isolation and purification procedure for chloroperoxidase isoenzymes

A simple four-step procedure has been developed for isolation of chloroperoxidase from the mold Caldariomyces fumago. Polyethyleneglycol precipitation of the contaminating pigment in the growth medium, followed by chromatography of the soluble enzyme fraction on a QAE-ZetaPrep-250 cartridge and ammonium sulfate precipitation affords isolation of the chloroperoxidase. Extensive dialysis and chromatography on DE-53 cellulose allows the separation and further purification of chloroperoxidase A and B isoenzymes.