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Polyphenol oxidases catalyze the hydroxylation of monophenols to diphenols, which are reducing agents for lytic polysaccharide monooxygenases (LPMOs) in their degradation of cellulose. In particular, the polyphenol oxidase MtPPO7 from Myceliophthora thermophila convert lignocellulose-derived monophenols, and under the new perspective of the peroxygenase reaction catalyzed by LPMOs, we aim to differentiate the role of the catalytic products of MtPPO7 in priming and fueling of LPMO activity. Exemplified by the activity of MtPPO7 towards guaiacol and by using the benchmark LPMO NcAA9C from Neurospora crassa we show that MtPPO7 catalytic products provide the initial electron for the reduction of Cu(II) to Cu(I) but cannot provide the required reducing power for continuous fueling of the LPMO. We show that the priming reaction occurs with catalytic amounts of MtPPO7 products and that those compounds do not generate substantial amounts of H2O2in situ to fuel the LPMO peroxygenase activity. Reducing agents with a low propensity to generate H2O2 can provide the means for controlling the LPMO catalysis through exogenous H2O2 and thereby minimize any enzyme inactivation.

Polyphenol oxidases catalyze the hydroxylation of monophenols to diphenols, which are reducing agents for lytic polysaccharide monooxygenases (LPMOs) in their degradation of cellulose. In particular, the polyphenol oxidase MtPPO7 from Myceliophthora thermophila converts lignocellulose-derived monophenols, and under the new perspective of the peroxygenase reaction catalyzed by LPMOs, we aim to differentiate the role of the catalytic products of MtPPO7 in priming and fueling of LPMO activity. Exemplified by the activity of MtPPO7 towards guaiacol and by using the benchmark LPMO NcAA9C from Neurospora crassa we show that MtPPO7 catalytic products provide the initial electron for the reduction of Cu(II) to Cu(I) but cannot provide the required reducing power for continuous fueling of the LPMO. The priming reaction is shown to occur with catalytic amounts of MtPPO7 products and those compounds do not generate substantial amounts of H2O2 in situ to fuel the LPMO peroxygenase activity. Reducing agents with a low propensity to generate H2O2 can provide the means for controlling the LPMO catalysis through exogenous H2O2 and thereby minimize any enzyme inactivation.

Copper-dependent lytic polysaccharide monooxygenases (LPMOs) classified in Auxiliary Activity (AA) families are considered indispensable as synergistic partners for cellulolytic enzymes to saccharify recalcitrant lignocellulosic plant biomass. In this study, we characterized two fungal oxidoreductases from the new AA16 family. We found that MtAA16A from Myceliophthora thermophila and AnAA16A from Aspergillus nidulans did not catalyze the oxidative cleavage of oligo- and polysaccharides. Indeed, the MtAA16A crystal structure showed a fairly LPMO-typical histidine brace active site, but the cellulose-acting LPMO-typical flat aromatic surface parallel to the histidine brace region was lacking. Further, we showed that both AA16 proteins are able to oxidize low-molecular-weight reductants to produce H2O2. The oxidase activity of the AA16s substantially boosted cellulose degradation by four AA9 LPMOs from M. thermophila (MtLPMO9s) but not by three AA9 LPMOs from Neurospora crassa (NcLPMO9s). The interplay with MtLPMO9s is explained by the H2O2-producing capability of the AA16s, which, in the presence of cellulose, allows the MtLPMO9s to optimally drive their peroxygenase activity. Replacement of MtAA16A by glucose oxidase (AnGOX) with the same H2O2-producing activity could only achieve less than 50% of the boosting effect achieved by MtAA16A, and earlier MtLPMO9B inactivation (6 h) was observed. To explain these results, we hypothesized that the delivery of AA16-produced H2O2to the MtLPMO9s is facilitated by protein-protein interaction. Our findings provide new insights into the functions of copper-dependent enzymes and contribute to a further understanding of the interplay of oxidative enzymes within fungal systems to degrade lignocellulose.

BACKGROUND: Arabinoxylan is the main fiber component in corn and corn co-products that are commonly included in pig diets. However, this fiber fraction is resistant to enzymatic degradation in the gastrointestinal tract of pigs. Ferulic acid and p-coumaric acid are covalently linked to arabinoxylan, so it is likely that the majority of these hydroxycinnamic acids are excreted in feces. However, data to confirm this have not been reported. The objective of this research was therefore to quantify the ferulic and p-coumaric acids in a diet based on corn and soybean meal (SBM) and in a diet based on corn, SBM, and distillers’ dried grains with solubles, as well as in feces from pigs fed these diets. RESULTS: The concentration of bound ferulic and coumaric acids in diets was greater in the corn-SBM-DDGS diet and in feces from pigs fed this diet than in the corn-SBM diet and feces from pigs fed that diet. The disappearance of free coumaric acids was greater (>85%) than that of bound phenolic acids (<50%) in both diets. The disappearance of free coumaric acid and bound ferulic acid in the intestinal tract of pigs was not different between the two diets. In contrast, disappearance of bound coumaric acid was greater (P < 0.05) in the corn-SBM diet than in the corn-SBM-DDGS diet. CONCLUSION: A diet based on corn and SBM contains less hydroxycinnamic acid than a corn-SBM-DDGS diet but bound phenolic acids are more resistant to digestion by pigs than free phenolic acids.