The coprophilous mushroom Coprinus radians secretes a haloperoxidase that catalyzes aromatic peroxygenation

Abstract

Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H2O2 concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent Km values for ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H2O2 were 49, 342, 635, 88, and 1,201 microM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.

FIG. 1.

(A) Effects of different amounts of soybean meal and glucose on peroxidase production by Coprinus radians DSM 888 in surface culture (data points represent maximum levels obtained within a cultivation period of 3 weeks). (B) Time course of peroxidase production by C. radians DSMZ 888 in the presence of 3% soybean meal and 4% glucose in agitated culture. Data points represent mean values for three parallel cultures (standard deviations, <10%). The dotted line marks the time course of pH. VA, veratryl alcohol.

FIG. 2.

FPLC elution profiles of CrP I (A) and CrP II and III (B) from Coprinus radians DSMZ 888. Separation was performed on a Mono Q column. Absorption at 405 nm (solid line), CrP activity detected by the oxidation of veratryl alcohol (VA) to veratraldehyde at pH 7 (•), and the NaCl gradient (dotted line) are shown.

FIG. 3.

Electrophoretic characterization of purified CrP isoforms. (A) SDS-PAGE of CrP II (lane 2), CrP III (lane 3), and CrP I (lane 4) after Mono Q separation. Lanes 1 and 5, protein standards; lane 6, CrP II after deglycosylation. (B) IEF of purified CrP I, CrP II, and CrP III after SEC separation. Lanes 7 and 8, CrP I; lane 9, CrP II; lane 10, CrP III; lane 11, protein standards.

FIG. 4.

N-terminal sequences (A) and peptide fragment alignments (B to E) of CrP II (Coprinus radians), AaP (Agrocybe aegerita), and CPO (Caldariomyces fumago). The numbering of amino acid residues was done on the basis of the known total sequence of CPO (30). (B) A peptide fragment of AaP consisting of 16 amino acids shows 71% identity to the sequence around the active site of CPO (positions 27 to 43) and includes the Cys₂₉ that is responsible for the binding of heme (fifth ligand [heme-thiolate]). (C) A second AaP fragment shows 27% identity. (D and E) Peptide sequences obtained for CrP II fragments showing 33% and 50% identities to the CPO sequence towards the C terminus.

FIG. 5.

UV-Vis absorption spectra of resting-state CrP II (4.6 μM) (thick line) and its reduced CO complex (thin line). The dotted line belongs to the spectrum of the dithionite-reduced enzyme.

FIG. 6.

Effects of pH on the CrP-catalyzed oxidation of veratryl alcohol (5 mM) (▴) and benzyl alcohol (5 mM) (•) (A) as well as DMP (2 mM) (⧫) and ABTS (0.6 mM) (▪) (B). Data points are means for three parallel measurements (standard deviations, <5%).

FIG. 7.

Bromination of phenol (A) and hydroxylation of naphthalene (B) by CrP II. HPLC elution profiles were recorded at 275 and 220 nm, respectively. (A) Peak 1, residual phenol; peak 2, 2-bromophenol; peak 3, 4-bromophenol. Insets show the UV spectra of 2-bromo- and 4-bromophenol. (B) Peak 1, residual naphthalene; peak 2, 1-naphthol; peak 3, 2-naphthol. The insets show the UV spectra of 1-naphthol (right) and 2-naphthol (left).