Characterization of the two Neurospora crassa cellobiose dehydrogenases and their connection to oxidative cellulose degradation.
The genome of Neurospora crassa encodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome of N. crassa and preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced in Pichia pastoris. With the cytochrome domain-dependent one-electron acceptor cytochrome c, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (k(cat) and K(m) values) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the heme b cofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) from N. crassa was expressed in P. pastoris. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.
Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.
Christoph
C
Sygmund
Daniel
D
Kracher
Stefan
S
Scheiblbrandner
Kawah
K
Zahma
Alfons K G
AK
Felice
Wolfgang
W
Harreither
Roman
R
Kittl
Roland
R
Ludwig
D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D002482:Cellulose; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase