Oxidation reactions catalyzed by manganese peroxidase isoenzymes from Ceriporiopsis subvermispora (original) (raw)
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Applied Biochemistry and Biotechnology, 2002
We have previously reported the oxidation of kojic acid catalyzed by manganese peroxidase (MnP) from Ceriporiopsis subvermispora. This reaction is strictly dependent on Mn(II), although it does not require the addition of hydrogen peroxide. We have extended these studies because this reaction can be considered as a model system for the in situ generation of hydrogen peroxide in natural environments. We show here that oxidation of kojic acid with horseradish peroxidase (HRP) plus hydrogen peroxide or with manganic acetate rendered a product with identical chromatographic and spectral properties as the one obtained in the reaction catalyzed by MnP. The initial lag observed in the latter reaction decreased significantly upon UV irradiation of the substrate. On the other hand, ascorbic acid increased the lag and did not affect the yield of the reaction. The superoxide anion trapping agents glutathione, nitroblue tetrazolium, and superoxide dismutase markedly affected the reaction. In contrast, addition of the hydroxyl radical scavengers mannitol and salicylic acid had no effect. Based on these results, a mechanism for the MnP-catalyzed reaction is proposed.
Applied and Environmental Microbiology, 2001
We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the ␣-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.
Fungal Genetics and Biology, 2010
The ligninolytic machinery of the widely used model fungus Ceriporiopsis subvermispora includes the enzymes manganese-peroxidase (MnP) and laccase (Lcs). In this work the effect of Mn(II) on the secretion of MnP was studied. Cultures grown in the absence of Mn(II) showed high levels of mnp transcripts. However, almost no MnP enzyme was detected in the extracellular medium, either by enzymatic activity assays or Western blot hybridizations. In the corresponding mycelia, immuno-electron microscopy experiments showed high levels of MnP enzyme within intracellular compartments. These results suggest that in addition to its well-known effect on transcription regulation of mnp genes, manganese influences secretion of MnP to the extracellular medium. Experiments carried out in the presence of cycloheximide confirmed that the metal is required to secrete MnP already synthesized and retained within the cell.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1996
Two manganese peroxidase isozymes, MnP1 and MnP2, were purified from the extracellular medium of ligninolytic cultures of Dichomitus squalens. The proteins were purified to homogeneity using DEAE-Sepharose chromatography and Mono Q fast protein liquid chromatography. MnP1 and MnP2 have molecular masses of 48 000 and 48 900 Da, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both isozymes are glycoproteins and each contains one iron protoporphyrin IX as a prosthetic group. The pl values of MnP1 and MnP2 are 4.15 and 3.90, respectively. N-Terminal amino-acid analysis suggests that these proteins are encoded by distinct genes. The Soret bands of the native ferric enzymes (408 nm and 406 nm, respectively) are shifted to 434 nm in the reduced enzymes and to 422 Jam in the reduced-CO complexes. EPR g-values of the native enzymes are essentially identical to those for other MnPs and lignin peroxidases, and they confirm the high-spin state of the iron. The addition of 1 equivalent of H202 to either of the native ferric isozymes yields spectra which are characteristic of compound I. Successive additions of 1 equivalent of ferrocyanide and 1 equivalent of H202 to the native enzymes yield spectra which are characteristic of compound II. Both MnP isozymes oxidize Mn 2+ to Mn 3 + in the presence of organic acid chelators. The MnP isozymes are produced by D. squalens only when the cells are grown in the presence of Mn.
Journal of Biotechnology, 2000
Two manganese-oxidizing peroxidases differing in glycosylation degree were purified from fermenter cultures of Bjerkandera sp. They were characterized and compared with the three manganese-oxidizing peroxidase isoenzymes obtained from the well-known ligninolytic fungus Phanerochaete chrysosporium. All the enzymes showed similar molecular masses but those from P. chrysosporium had less acidic isoelectric point. Moreover, the latter strictly required Mn 2 + to oxidize phenolic substrates whereas the Bjerkandera peroxidases had both Mn-mediated and Mn-independent activity on phenolic and non-phenolic aromatic substrates. Taking into account these results, and those reported for Bjerkandera adusta and different Pleurotus species, we concluded that two different types of Mn 2 +-oxidizing peroxidases are secreted by ligninolytic fungi.
Enzyme and Microbial Technology, 2002
Extracellular manganese peroxidase (MnP) was purified from liquid cultures of the litter-decomposing basidiomycetes Agrocybe praecox and Stropharia coronilla. Both fungi produced MnP increasingly in response to Mn 2ϩ in the medium. A. praecox secreted two MnP isoforms with similar isoelectric points (pI) of 6.3-7.0 and a molecular weight (MW) of 42 kDa. MnP activity was not observed in Mn 2ϩ -free cultures of A. praecox. In Mn 2ϩ -supplemented cultures, S. coronilla produced at least two MnPs, of which the main isoform MnP1 has a pI of 6.3-7.1 and a MW of 41 kDa. In addition, S. coronilla possesses a partly constitutive MnP (MnP2) which was also detectable in Mn 2ϩ -free cultures, although its amount was considerably lower. MnP2 showed two distinct bands with acidic pIs of 3.5 and 3.7 in the IEF gel and has a MW of 41 kDa. There are indications for the existence of a third, likewise Mn 2ϩ -inducible enzyme (MnP3), that could not be separated from MnP2 but formed an additional band in eletrophoretic analyses (pI 5.1, MW 43 kDa).
Archives of Biochemistry and Biophysics, 1998
The kinetics of Mn 3+-oxalate formation and decay were investigated in reactions catalyzed by manganese peroxidase (MnP) from the basiomycete Ceriporiopsis subvermispora in the absence of externally added hydrogen peroxide. A characteristic lag observed in the formation of this complex was shortened by glyoxylate or catalytic amounts of Mn 3+ or hydrogen peroxide. MnP titers had a minor effect on this lag and did not influence the decay rate of the complex. In contrast, Mn 2+ and oxalate drastically affected maximal concentrations of the Mn 3+-oxalate complex formed, the decay of which was accelerated at high Mn 2+ levels. The highest concentration of complex was obtained at pH 4.0, whereas an inverse relationship was found between the pH of the reaction and the decay rate of the complex with MnP present. In the absence of MnP, the best fit for the decay kinetics of the complex gave an order of 3/2 at concentrations in the range of 30-100 µM, with a k obs = 0.012 min-1 M-0.5 at pH 4.0. The rate constant increases at lower pH values and decreases at high oxalate concentrations. The physiological relevance of these findings is discaused.
Current Genetics, 2003
We previously identified and characterized three mnp genes coding for manganese peroxidase (MnP) in the white rot fungus Ceriporiopsis subvermispora. In this work, we assessed transcript levels of mnp genes in liquid cultures of this fungus grown under various conditions. In the absence of Mn 2+ , mnp1 and mnp2 mRNA were detected by Northern hybridization, irrespective of the lack of extracellular MnP activity. Addition of Mn 2+ to the cultures led to a marked increase in both transcripts, the highest titers being observed at 10 lM Mn 2+ . mnp1 mRNA was not detected at Mn 2+ concentrations above 80 lM, whereas mnp2 mRNA was still observed at 320 lM Mn 2+ . Differential regulation of these genes was confirmed by the addition of Cu 2+ , Zn 2+ , Ag + and Cd 2+ . These metal ions dramatically elevated both transcripts and also allowed the detection of the mnp3 transcript. In most cases, the increase in mRNA levels was partially abolished by the simultaneous presence of Mn 2+ , although the latter was strictly required to detect extracellular MnP activity. However, the lignin-related compound syringic acid specifically increased the mnp1 transcript, although only in the absence of Mn 2+ . These results indicate that there is no clear correlation between mnp mRNA levels and MnP activity. In addition, they strongly suggest that Mn 2+ plays a post-transcriptional role which is essential for the presence of active MnP in the extracellular fluid.
Purification and Catalytic Properties of Two Manganese Peroxidase Isoenzymes from Pleurotus eryngii
European Journal of Biochemistry, 1996
The ligninolytic basidiomycetes Pleurotus eryngii, Pleurotus ostreatus, Pleurotus pulmonarius and Pleurotus sajor-caju did not exhibit detectable levels of manganese peroxidase (MP) when grown in liquid media with ammonium tartrate as N source. However, after examination of cells grown on different organic N-based media, high M P activity was obtained in peptone medium, up to nearly 3 U/ml in cultures of I? eryngii. Moreover, Mnz+ supplementation was not used to produce MP, since all Mn2+ concentrations assayed (1 -4000 pM) inhibited production of this enzyme in liquid medium.
Description of the first fungal dye-decolorizing peroxidase oxidizing manganese(II)
Applied Microbiology and Biotechnology, 2015
Two phylogenetically divergent genes of the new family of dye-decolorizing peroxidases (DyPs) were found during comparison of the four DyP genes identified in the Pleurotus ostreatus genome with over 200 DyP genes from other basidiomycete genomes. The heterologously expressed enzymes (Pleos-DyP1 and Pleos-DyP4, following the genome nomenclature) efficiently oxidize anthraquinoid dyes (such as Reactive Blue 19), which are characteristic DyP substrates, as well as low redox-potential dyes (such as 2,2azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and substituted phenols. However, only Pleos-DyP4 oxidizes the high redox-potential dye Reactive Black 5, at the same time that it displays high thermal and pH stability. Unexpectedly, both enzymes also oxidize Mn 2+ to Mn 3+ , albeit with very different catalytic efficiencies. Pleos-DyP4 presents a Mn 2+ turnover (56 s −1) nearly in the same order of the two other Mn 2+-oxidizing peroxidase families identified in the P. ostreatus genome: manganese peroxidases (100 s −1 average turnover) and versatile peroxidases (145 s −1 average turnover), whose genes were also heterologously expressed. Oxidation of Mn 2+ has been reported for an Amycolatopsis DyP (24 s −1) and claimed for other bacterial DyPs, albeit with lower activities, but this is the first time that Mn 2+ oxidation is reported for a fungal DyP. Interestingly, Pleos-DyP4 (together with ligninolytic peroxidases) is detected in the secretome of P. ostreatus grown on different lignocellulosic substrates. It is suggested that generation of Mn 3+ oxidizers plays a role in the P. ostreatus white-rot lifestyle since three different families of Mn 2+-oxidizing peroxidase genes are present in its genome being expressed during lignocellulose degradation.