Purification and characterization of two manganese peroxidase isozymes from the white-rot basidiomycete Dichomitus squalens.
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 48000 and 48900 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 nm 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 H2O2 to either of the native ferric isozymes yields spectra which are characteristic of compound 1. Successive additions of 1 equivalent of ferrocyanide and 1 equivalent of H2O2 to the native enzymes yield spectra which are characteristic of compound II. Both MnP isozymes oxidize Mn2+ to Mn3+ 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.
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Department of Chemistry, Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.
Sheng
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D001487:Basidiomycota; D015153:Blotting, Western; D002614:Chelating Agents / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D017124:Conserved Sequence / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D010544:Peroxidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity
D002614:Chelating Agents; D005656:Fungal Proteins; D007527:Isoenzymes; D008670:Metals; D008345:Manganese; D010544:Peroxidases; C051129:manganese peroxidase