Characterization of the two Neurospora crassa cellobiose dehydrogenases and their connection to oxidative cellulose degradation

Appl Environ Microbiol. 2012 Sep;78(17):6161-71. doi: 10.1128/AEM.01503-12. Epub 2012 Jun 22.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbohydrate Dehydrogenases / chemistry
  • Carbohydrate Dehydrogenases / genetics
  • Carbohydrate Dehydrogenases / metabolism*
  • Cellulose / metabolism*
  • Enzyme Stability
  • Hydrogen-Ion Concentration
  • Kinetics
  • Neurospora crassa / enzymology*
  • Neurospora crassa / genetics
  • Neurospora crassa / metabolism*
  • Oxidation-Reduction
  • Pichia / genetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • Recombinant Proteins
  • Cellulose
  • Carbohydrate Dehydrogenases
  • cellobiose-quinone oxidoreductase