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

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.

Fig 1

Production of recombinant CDH IIA (A), CDH IIB (B), and GH61-3 (C) in P. pastoris. Black circles, wet biomass; gray triangles, volumetric activity (DCIP assay at pH 5.0); black diamonds, extracellular protein concentration (Bradford assay). The measurements were done in duplicates; the difference between the values was <5%.

Fig 2

(A and B) Spectral characterization of CDH IIA (A) and CDH IIB (B) showing the oxidized (black) and reduced (gray) spectra. Lactose was used for reduction. The difference spectra (oxidized-reduced) are given in the insets. (C) Spectrum of oxidized GH61-3, with the inset showing the spectrum of the type 2 copper atom in its oxidized (black) and reduced (gray) states. Ascorbate was used to reduce the enzyme.

Fig 3

Electrochemical measurements of both CDHs on thiol-modified gold electrodes at pH 6.0 (A) and pH 7.5 (B). Midpoint potentials of both CDHs were calculated from cyclic voltammograms (left) and square-wave voltammograms (middle) in the absence of a substrate. The cyclic voltammetry (right) in the presence of the substrate was used to measure the current of direct electron transfer to a gold electrode.

Fig 4

(A to D) pH dependency of CDH IIA and CDH IIB activities for the artificial electron acceptors ferrocenium hexafluorophosphate (A), cytochrome c (B), 1,4-benzoquinone (C), and DCIP (D), using lactose as the electron donor. Circles, CDH IIA; triangles, CDH IIB. Activities from pH 2.5 to 9 were measured with McIlvaine buffer. (E and F) pH-dependent interaction of CDH IIA (E) and CDH IIB (F) with the competitive substrates cyt c and GH61-3. Diamonds, turnover rates of cyt c in the absence of GH61-3; squares, turnover rates of cyt c in the presence of GH61-3; filled circles, ratio of the turnover numbers, which is used as a measure of the inhibition of cyt c reduction by GH61-3.

Fig 5

Phylogenetic tree of the N. crassa GH61 protein family. Sequence alignment was performed with Clustal using the default parameters, and phylogenetic analysis was done with MEGA 5 using the maximum likelihood method and 1,000 bootstrap replicates. Relative expression levels of N. crassa gh61 genes during growth on Miscanthus (16 h) are shown. Data are taken from data reported previously by Tian et al. (32). n.d., not determined.