The interplay between lytic polysaccharide monooxygenases and glycoside hydrolases.

In nature, enzymatic degradation of recalcitrant polysaccharides such as chitin and cellulose takes place by a synergistic interaction between glycoside hydrolases (GHs) and lytic polysaccharide monooxygenases (LPMOs). The two different families of carbohydrate-active enzymes use two different mechanisms when breaking glycosidic bonds between sugar moieties. GHs employ a hydrolytic activity and LPMOs are oxidative. Consequently, the topologies of the active sites differ dramatically. GHs have tunnels or clefts lined with a sheet of aromatic amino acid residues accommodating single polymer chains being threaded into the active site. LPMOs are adapted to bind to the flat crystalline surfaces of chitin and cellulose. It is believed that the LPMO oxidative mechanism provides new chain ends that the GHs can attach to and degrade, often in a processive manner. Indeed, there are many reports of synergies as well as rate enhancements when LPMOs are applied in concert with GHs. Still, these enhancements vary in magnitude with respect to the nature of the GH and the LPMO. Moreover, impediment of GH catalysis is also observed. In the present review, we discuss central works where the interplay between LPMOs and GHs has been studied and comment on future challenges to be addressed to fully use the potential of this interplay to improve enzymatic polysaccharide degradation.

A comparative biochemical investigation of the impeding effect of C1-oxidizing LPMOs on cellobiohydrolases.

Lytic polysaccharide monooxygenases (LPMOs) are known to act synergistically with glycoside hydrolases in industrial cellulolytic cocktails. However, a few studies have reported severe impeding effects of C1-oxidizing LPMOs on the activity of reducing-end cellobiohydrolases. The mechanism for this effect remains unknown, but it may have important implications as reducing-end cellobiohydrolases make up a significant part of such cocktails. To elucidate whether the impeding effect is general for different reducing-end cellobiohydrolases and study the underlying mechanism, we conducted a comparative biochemical investigation of the cooperation between a C1-oxidizing LPMO from Thielavia terrestris and three reducing-end cellobiohydrolases; Trichoderma reesei (TrCel7A), T. terrestris (TtCel7A), and Myceliophthora heterothallica (MhCel7A). The enzymes were heterologously expressed in the same organism and thoroughly characterized biochemically. The data showed distinct differences in synergistic effects between the LPMO and the cellobiohydrolases; TrCel7A was severely impeded, TtCel7A was moderately impeded, while MhCel7A was slightly boosted by the LPMO. We investigated effects of C1-oxidations on cellulose chains on the activity of the cellobiohydrolases and found reduced activity against oxidized cellulose in steady-state and pre-steady-state experiments. The oxidations led to reduced maximal velocity of the cellobiohydrolases and reduced rates of substrate complexation. The extent of these effects differed for the cellobiohydrolases and scaled with the extent of the impeding effect observed in the synergy experiments. Based on these results, we suggest that C1-oxidized chain ends are poor attack sites for reducing-end cellobiohydrolases. The severity of the impeding effects varied considerably among the cellobiohydrolases, which may be relevant to consider for optimization of industrial cocktails.