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Characterization of Carbohydrate-Binding Cytochrome b₅₆₂ from the White-Rot Fungus Phanerochaete chrysosporium

Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan,¹ National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan,² Department of Biotechnology, Tokyo University of Agricultural and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan³

Received 29 October 2004/ Accepted 18 February 2005

cDNA encoding a hemoprotein similar to the cytochrome domain of extracellular flavocytochrome cellobiose dehydrogenase (CDH) was cloned from the white-rot fungus Phanerochaete chrysosporium. The deduced amino acid sequence implies that there is a two-domain structure consisting of an N-terminal cytochrome domain and a C-terminal family 1 carbohydrate-binding module (CBM1) but that the flavin-containing domain of CDH is not present. The gene transcripts were observed in cultures in cellulose medium but not in cultures in glucose medium, suggesting that there is regulation by carbon catabolite repression. The gene was successfully overexpressed in Pichia pastoris, and the recombinant protein was designated carbohydrate-binding cytochrome b₅₆₂ (CBCyt. b₅₆₂). The resonance Raman spectrum suggested that the heme of CBCyt. b₅₆₂ is 6-coordinated in both the ferric and ferrous states. Moreover, the redox potential measured by cyclic voltammetry was similar to that of the cytochrome domain of CDH. These results suggest that the redox characteristics may be similar to those of the cytochrome domain of CDH, and so CBCyt. b₅₆₂ may have an electron transfer function. In a binding study with various carbohydrates, CBCyt. b₅₆₂ was adsorbed with high affinity on both cellulose and chitin. As far as we know, this is the first example of a CBM1 connected to a domain without apparent catalytic activity for carbohydrate; this CBM1 may play a role in localization of the redox protein on the surface of cellulose or on the fungal sheath in vivo.

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