Cellobiose Dehydrogenase from the Fungi Phanerochaete chrysosporium and Humicola insolens

Abstract

Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporium and Humicola insolens. The pH optimum of the cellobiose-cytochrome c oxidoreductase activity of P. chrysosporium CDH was acidic, whereas that of H. insolens CDH was neutral. The absorption spectra of the two CDHs showed them to be typical hemoproteins, but there was a small difference in the visible region. Limited proteolysis between the heme and flavin domains was performed to investigate the cofactors. There was no difference in absorption spectrum between the heme domains of P. chrysosporium and H. insolens CDHs. The midpoint potentials of heme at pH 7.0 were almost identical, and no difference in pH dependence was observed over the range of pH 3-9. The pH dependence of cellobiose oxidation by the flavin domains was similar to that of the native CDHs, indicating that the difference in the pH dependence of the catalytic activity between the two CDHs is because of the flavin domains. The absorption spectrum of the flavin domain from H. insolens CDH has absorbance maxima at 343 and 426 and a broad absorption peak at 660 nm, whereas that of P. chrysosporium CDH showed a normal flavoprotein spectrum. Flavin cofactors were extracted from the flavin domains and analyzed by high-performance liquid chromatography. The flavin cofactor from H. insolens was found to be a mixture of 60% 6-hydroxy-FAD and 40% FAD, whereas that from P. chrysosporium CDH was normal FAD. After reconstitution of the deflavo-proteins it was found that flavin domains containing 6-hydroxy-FAD were clearly active but their cellobiose oxidation rates were lower than those of flavin domains containing normal FAD. Reconstitution of flavin cofactor had no effect on the optimum pH. From these results, it is concluded that the pH dependence is not because of the flavin cofactor but is because of the protein molecule.

Biophysical and Structural Analysis of a Novel Heme b Iron Ligation in the Flavocytochrome Cellobiose Dehydrogenase

“…4 and Table II). This value was in close agreement with previous electrochemical measurements of native CDH (37) and its truncated heme domain (38) and similar to the value of the heme group in cytochrome b 562 (39), a second example of a b-type heme with a Met-His coordination. Substitution of Met 65 by histidine resulted in a 210-mV drop to Ϫ53 mV (Fig.…”

Cellobiose Dehydrogenase – A Flavocytochrome from Wood-Degrading, Phytopathogenic and Saprotropic Fungi

“…The flavin cofactor of H. insolens CDH was found to be a mixture of 60% 6-hydroxysubstituted flavin adenine dinucleotide (6-OH FAD) and 40% FAD. Dehydrogenase domains carrying 6-OH FAD exhibited lower rates of cellobiose oxidation [38]. A truly thermophilic CDH from Sporotrichum thermophile [39, synonym Thielavia heterothallica] exhibited a temperature optimum of 60°C.…”

The Human Apoptosis-inducing Protein AMID Is an Oxidoreductase with a Modified Flavin Cofactor and DNA Binding Activity

“…A number of enzymes have now been purified in which the FAD cofactor exists partially in a form that is hydroxylated at C-6 of the isoalloxazine ring (20,22,(25)(26)(27)(28). In many cases, this yields a catalytically inactive enzyme or one with reduced activity compared with the FAD-containing protein and represents only a small percentage of the total flavin content.…”