ABSTRACT
The lignin peroxidase (LIP) isozyme profile of the white-rot fungus Phanerochaete chrysosporium changes markedly with culture age. This change occurs extracellularly and results from enzymatic dephosphorylation of LIP isozymes. In this study, a novel mannose 6-phosphatase (M6Pase) from extracellular culture fluid filtrate of P. chrysosporium, shown to be responsible for the extracellular postranslational modification of LIP, was purified and characterized. In vitro incubation of the purified M6Pase with purified LIP isozyme H2 resulted in its conversion to isozyme H1, with an equimolar release of orthophosphate. Using different sugar phosphates as substrate, the enzyme exhibited narrow specificity, showing activity mostly for mannose 6-phosphate (K(m) = 0.483 mM). The enzyme displayed a molecular mass of 82 kDa, as determined by gel filtration, and 40.4 and 39.1 kDa, on SDS-PAGE, suggesting that the native form is a dimer. The N-terminal sequence of the enzyme has no homology with that of other reported phosphatases. M6Pase is a metalloprotein with manganese and cobalt as the preferred metal ions. It is N-glycosylated proteins with an isoelectric point of 4. 7-4.8 and a pH optimum of 5. Based on its characteristics, M6Pase from P. chrysosporium seems to be a unique phosphatase responsible for posttranslation modification of LIP isozymes.
Subject(s)
Peroxidases/metabolism , Phanerochaete/enzymology , Phosphoric Monoester Hydrolases/metabolism , Amino Acid Sequence , Hydrogen-Ion Concentration , Isoelectric Point , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Metals/pharmacology , Molecular Sequence Data , Molecular Weight , Peroxidases/isolation & purification , Phanerochaete/genetics , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/genetics , Phosphorylation , Protein Processing, Post-Translational , Substrate SpecificityABSTRACT
The combined effects of Mn and oxygen on lignin peroxidase (LIP) activity and isozyme composition in Phanerochaete chrysosporium were studied by using shallow stationary cultures grown in the presence of limited or excess N. When no Mn was added, LIP was formed in both N-limited and N-excess cultures exposed to air, but no LIP activity was observed at Mn concentrations greater than 13 mg/liter. In oxygen-flushed, N-excess cultures, LIP was formed at all Mn concentrations, and the peak LIP activity values in the extracellular fluid were nearly identical in the presence of Mn concentrations ranging from 3 to 1,500 mg/liter. When the availability of oxygen to cultures exposed to air was increased by growing the fungus under nonimmersed liquid conditions, higher levels of Mn were needed to suppress LIP formation compared with the levels needed in shallow stationary cultures. The composition of LIP isozymes was affected by the levels of N and Mn. Addition of veratryl alcohol to cultures exposed to air did not eliminate the suppressive effect of Mn on LIP formation. A deficiency of Mn in N-excess cultures resulted in lower biomass and a lower rate of glucose consumption than in the presence of Mn. In addition, almost no activity of the antioxidant enzyme Mn superoxide dismutase was observed in Mn-deficient, N-excess cultures, but the activity of this enzyme increased as the Mn concentration increased from 3 to 13 mg/liter. No Zn/Cu superoxide dismutase activity was observed in N-excess cultures regardless of the Mn concentration.
