Novel interaction between laccase and cellobiose dehydrogenase during pigment synthesis in the white rot fungus Pycnoporus cinnabarinus

When glucose is the carbon source, the white rot fungus Pycnoporus cinnabar inus produces a characteristic red pigment, cinnabarinic acid, which is for med by laccase-catalyzed oxidation of the precursor 3-hydroxyanthranilic ac id. When P. cinnabarinus was grown on media containing cellobiose or cellul ose as the carbon source, the amount of cinnabarinic acid that accumulated was reduced or, in the case of cellulose, no cinnabarinic acid accumulated. Cellobiose-dependent quinone reducing enzymes, the cellobiose dehydrogenas es (CDHs), inhibited the redox interaction between laccase and 3-hydroxyant hranilic acid. Two distinct proteins were purified from cellulose-grown cul tures of P. cinnabarinus; these proteins were designated CDH I and CDH II. CDH I and CDH II were both monomeric proteins and had apparent molecular we ights of about 81,000 and 101,000, respectively, as determined by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T he pi values were approximately 5.9 for CDH I and 3.8 for CDH II. Both CDHs used several known CDH substrates as electron accepters and specifically a dsorbed to cellulose. Only CDH II could reduce cytochrome c. The optimum pH values fbr CDH I and CDH II were 5.5 and 4.5, respectively. In in vitro ex periments, both enzymes inhibited laccase-mediated formation of cinnabarini c acid. Oxidation intermediates of 3-hydroxyanthranilic acid served as endo genous electron accepters for the two CDHs from P. cinnabarinus. These resu lts demonstrated that in the presence of a suitable cellulose-derived elect ron donor, CDHs can regenerate fungal metabolites oxidized by laccase, and they also supported the hypothesis that CDHs act as links between celluloly tic and ligninolytic pathways.