Characterization of carbohydrate-binding cytochrome b562 from the white-rot fungus Phanerochaete chrysosporium.
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.
Yoshida
Makoto
M
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.
Igarashi
Kiyohiko
K
Wada
Masahisa
M
Kaneko
Satoshi
S
Suzuki
Norio
N
Matsumura
Hirotoshi
H
Nakamura
Nobuhumi
N
Ohno
Hiroyuki
H
Samejima
Masahiro
M
D000595:Amino Acid Sequence; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000378:metabolism*; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D003573:Cytochrome b Group; D018076:DNA, Complementary; D011994:Recombinant Proteins; D002482:Cellulose; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase