Fig. 2. Occurrence and analysis of oxidoreductases linked to lignocellulose degradation. (A) Relative occurrence of cdh, lpmo, and gmc (cdh excluded) genes in 97 genomes grouped by fungal lifestyles. The relative occurrence is indicated by color-coded bars, normalized to the number (n) of analyzed genomes given above [cdh and lpmo genes, red; gmc (cdh excluded) and lpmo genes, yellow; only lpmo genes, blue; none of these, gray]. None of the genomes contained cdh without lpmo. Numbers in the bars indicate the average number of lpmo genes for fungi in each category. The lifestyle of a fungus is often uncertain, and published data allowed the assignment of only 81 genomes (table S1).
(fig. S5) but much higher in genomes that also contained cdh. The latter genomes contained between 9 and 23 lpmo genes on average, which indicates the presence of a diversified set of LPMOs that are likely to carry out a range of catalytic tasks. The factor map generated by principal component analysis shows a high correlation between the LPMO and CDH variables
(Fig. 2B and fig. S6). The clustering of individual data sets corresponds to the reported lifestyles for most of the investigated fungal species. The clusters of brown rots and plant symbionts correlate negatively with all extracellular oxidoreductase variables. The loss of oxidoreductases in brown rots has been explained by an evolutionary contraction (16).
Fig. 3. CDH-catalyzed LPMO reduction. (A) Electron transfer rates from N. crassa CDH IIA to LPMOs were measured by stopped-flow spectroscopy. LPMOs with different oxidative regioselectivity and cellulose-binding properties were tested (LPMO-02916, which has a CBM1, oxidizes cellulose at position C4; LPMO-01867 and LPMO-08760 oxidize at C1 and have a CBM1; LPMO-03328 oxidizes at C1 and lacks a CBM1). The observed heme b oxidation rates (0.9 to 20.6 s−1) at pH 6.0 were much higher than the reoxidation rate with oxygen (100 to 2100 times as high; table S6) or ferric iron (30 to 660 times as high; table S7). (B) The pH dependency of the observed electron transfer rates from CDH IIA or CDH IIB to each of the four LPMOs [same color code as in (A)] indicates a slightly acidic
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(B) Principal component analysis. The numbers of genes encoding CDH, GMC (CDH excluded), LPMO (CAZy family AA9), cellulase (Cel), laccase (Lac), and ligninolytic activities (POD) per genome were used as variables (small symbols; large symbols indicate group-specific centers of gravity). A high correlation among the analyzed genes is indicated by a similar orientation of the dark blue vectors.The contribution of the variables to the overall data variance is indicated by the line style of the vectors. The individual contribution (percent) to each dimension is further specified in the axis legend. Outliers in the lifestyle-dependent clustering of individual data sets could originate from an incorrect assignment.
CDH is a reported activator of LPMO (11, 13). To investigate the catalytic efficiency of CDH and thus the biological relevance of LPMO-CDH cooccurrence, the interactions of both enzymes were tested by rapid spectroscopy. The Neurospora crassa genome has two cdh and 14 lpmo genes, and both CDHs [CDH IIA, carrying a family 1 carbohydrate binding module (CBM1), and CDH IIB, lacking
optimum pH. LPMO-08760 shows a preference for CDH IIA as an electron donor, whereas LPMO-02916 and LPMO-01867 were reduced faster by CDH IIB. Circles represent mean values of at least three replicates, and error bars represent standard deviations. (C and D) Electron transfer from a glassy carbon electrode to LPMO, mediated by (C) CDH IIA or (D) its isolated cytochrome domain (I, current; E, redox potential). Cyclic voltammetry showed a charging current for LPMO in the absence of oxygen; the onset potential is limited by the reduction potential of the heme b cofactor. In the presence of oxygen, a catalytic current was observed. The higher current observed for the cytochrome domain–mediated process may be due to its higher mobility compared with that of CDH. Data for LPMO-02916 are presented in fig. S9. 27 MAY 2016 • VOL 352 ISSUE 6289
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