The Pyrroloquinoline-Quinone-Dependent Pyranose Dehydrogenase from Coprinopsis cinerea Drives Lytic Polysaccharide Monooxygenase Action

Abstract

ABSTRACT Fungi secrete a set of glycoside hydrolases and oxidoreductases, including lytic polysaccharide monooxygenases (LPMOs), for the degradation of plant polysaccharides. LPMOs catalyze the oxidative cleavage of glycosidic bonds after activation by an external electron donor. So far, only flavin-dependent oxidoreductases (from the auxiliary activity [AA] family AA3) have been shown to activate LPMOs. Here, we present LPMO activation by a pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase (PDH) from Coprinopsis cinerea , Cc PDH, the founding member of the recently discovered auxiliary activity family AA12. Cc PDH contains a C-terminal family 1 carbohydrate binding module (CBM1), an N-terminal family AA8 cytochrome domain, and a central AA12 dehydrogenase domain. We have studied the ability of full-length Cc PDH and its truncated variants to drive catalysis by two Neurospora crassa LPMOs. The results show that Cc PDH indeed can activate the C-1-oxidizing N. crassa LPMO 9F ( Nc LPMO9F) and the C-4-oxidizing Neurospora crassa LPMO 9C ( Nc LPMO9C), that this activation depends on the cytochrome domain, and that the dehydrogenase and the LPMO reactions are strongly coupled. The two tested Cc PDH-LPMO systems showed quite different efficiencies, and this difference disappeared upon the addition of free PQQ acting as a diphenol/quinone redox mediator, showing that LPMOs differ when it comes to their direct interactions with the cytochrome domain. Surprisingly, removal of the CBM domain from Cc PDH had a considerable negative impact on the efficiency of the Cc PDH-LPMO systems, suggesting that electron transfer in the vicinity of the substrate is beneficial. Cc PDH does not oxidize cello-oligosaccharides, which makes this enzyme a useful tool for studying cellulose-oxidizing LPMOs. IMPORTANCE Lytic polysaccharide monooxygenases (LPMOs) are currently receiving increasing attention because of their importance in degrading recalcitrant polysaccharides and their potential roles in biological processes, such as bacterial virulence. LPMO action requires an external electron donor, and fungi growing on biomass secrete various so-called glucose-methanol-choline (GMC) oxidoreductases, including cellobiose dehydrogenase, which can donate electrons to LPMOs. This paper describes how an enzyme not belonging to the GMC oxidoreductase family, Cc PDH, can activate LPMOs, and it provides new insights into the activation process by (i) describing the roles of individual Cc PDH domains (a dehydrogenase, a cytochrome, and a carbohydrate-binding domain), (ii) showing that the PDH and LPMO enzyme reactions are strongly coupled, (iii) demonstrating that LPMOs differ in terms of their efficiencies of activation by the same activator, and (iv) providing indications that electron transferring close to the substrate surface is beneficial for the overall efficiency of the Cc PDH-LPMO system.