Kiyohiko Igarashi

Cellobiose Dehydrogenase from the Fungi Phanerochaete chrysosporium and Humicola insolens A FLAVOHEMOPROTEIN FROM HUMICOLA INSOLENS CONTAINS 6-HYDROXY-FAD AS THE DOMINANT ACTIVE COFACTOR

Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporium and Humicola insolens. The pH optimum of the cellobiose-cytochrome c oxidoreductase activity ofP. chrysosporium CDH was acidic, whereas that of H. insolens CDH was neutral. The absorption spectra of the two CDHs showed them to be typical hemoproteins, but there was a small difference in the visible region. Limited proteolysis between the heme and flavin domains was performed to investigate the cofactors. There was no difference in absorption spectrum between the heme domains ofP. chrysosporium and H. insolens CDHs. The midpoint potentials of heme at pH 7.0 were almost identical, and no difference in pH dependence was observed over the range of pH 3–9. The pH dependence of cellobiose oxidation by the flavin domains was similar to that of the native CDHs, indicating that the difference in the pH dependence of the catalytic activity between the two CDHs is because of the flavin domains. The absorption spectrum of the flavin domain fromH. insolens CDH has absorbance maxima at 343 and 426 and a broad absorption peak at 660 nm, whereas that of P. chrysosporium CDH showed a normal flavoprotein spectrum. Flavin cofactors were extracted from the flavin domains and analyzed by high-performance liquid chromatography. The flavin cofactor fromH. insolens was found to be a mixture of 60% 6-hydroxy-FAD and 40% FAD, whereas that from P. chrysosporium CDH was normal FAD. After reconstitution of the deflavo-proteins it was found that flavin domains containing 6-hydroxy-FAD were clearly active but their cellobiose oxidation rates were lower than those of flavin domains containing normal FAD. Reconstitution of flavin cofactor had no effect on the optimum pH. From these results, it is concluded that the pH dependence is not because of the flavin cofactor but is because of the protein molecule.

Kojima, Y., Várnai, A., Ishida, T., Sunagawa, N., Petrovic, D. M., Igarashi, K., ... & Eijsink, V. G. (2016). A lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown-rot fungus Gloeophyllum trabeum and its action on cellulose-xyloglucan complexes. Applied and environmental microbiology, 82(22), 6557-6572.

Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum , tend to have few LPMOs and information on these enzymes is scarce. The genome of G. trabeum encodes four AA9 LPMOs, whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of Gt LPMO9A seem to be produced, a single domain variant, Gt LPMO9A-1, and a longer variant, Gt LPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct Gt LPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses, using HPAEC-PAD and MS, showed that Gt LPMO9A-2 is active on cellulose, carboxymethylcellulose and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, Gt LPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of Gt LPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, Nc LPMO9C from Neurospora crassa , revealed that Gt LPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurments also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of Nc LPMO9C. Experiments with co-polymeric substrates showed an inhibitory effect of hemicellulose-coating on cellulolytic LPMO activity and did not reveal additional activities of Gt LPMO9A-2. These results provide insight into the LPMO-potential of G. trabeum and provide a novel sensitive method, measurement of dynamic viscosity, for monitoring LPMO activity. Importance Currently, there are only a few methods available to analyze end-products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here we present an alternative and sensitive method based on measurement of dynamic viscosity, for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses such as xyloglucan. We have used both this novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown rot fungus. This enzyme, Gt LPMO9A-2, differs from previously characterized LPMOs, in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. Gt LPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibres together. The xyloglucan-degrading potential of Gt LPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown-rot, through degradation of the primary cell wall.ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.