Phosphorylation of lignin peroxidases from Phanerochaete chrysosporium. Identification of mannose 6-phosphate.
Many of the extracellular lignin-degrading peroxidases from the wood-degrading fungus Phanerochaete chrysosporium are phosphorylated. Immunoprecipitation of the extracellular fluid of cultures grown with H2K32PO4 with a polyclonal antibody raised against one of the lignin peroxidase isozymes, H8 (pI 3.5), revealed the incorporation of H2K32PO4 into lignin peroxidases. Analyses of the purified isozymes from labeled cultures by isoelectric focusing showed that, in addition to isozyme H8, lignin peroxidase isozymes H2 (pI 4.4), H6 (pI 3.7), and H10 (pI 3.3) are also phosphorylated. These analyses also showed that lignin peroxidase isozyme H1 (pI 4.7) and manganese-dependent peroxidase isozymes H3 (pI 4.9) and H4 (pI 4.5) are not phosphorylated. Phosphate quantitation indicated the presence of one molecule of phosphate/molecule of enzyme for all of the phosphorylated isozymes. To locate the site of phosphorylation, one-dimensional phosphoamino acid analysis was performed with hydrolyzed 32P-protein. However, phosphotyrosine, phosphoserine, and phosphothreonine could not be identified. Coupled enzyme assays of acid hydrolysate indicated the presence of mannose 6-phosphate as the phosphorylated component on the lignin peroxidase isozymes. Digestion of the isozymes with N-glycanase released the phosphate component, indicating that the mannose 6-phosphate is contained on an asparagine-linked oligosaccharide.
Kuan
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IC
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Tien
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D000363:Agaricales / Q000201:enzymology*; D000596:Amino Acids / Q000032:analysis; D006600:Hexosephosphates / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008360:Mannosephosphates / Q000302:isolation & purification / Q000378:metabolism*; D010544:Peroxidases / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation
D000596:Amino Acids; D006600:Hexosephosphates; D007527:Isoenzymes; D008360:Mannosephosphates; C027693:mannose-6-phosphate; D010544:Peroxidases; C042858:lignin peroxidase