Abstract
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the oxidative cleavage of polysaccharides such as cellulose and chitin, a feature that makes them key tools in industrial biomass conversion processes. The catalytic domains of a considerable fraction of LPMOs and other carbohydrate-active enzymes (CAZymes) are tethered to carbohydrate-binding modules (CBMs) by flexible linkers. These linkers preclude X-ray crystallographic studies, and the functional implications of these modular assemblies remain partly unknown. Here, we used NMR spectroscopy to characterize structural and dynamic features of full-length modular ScLPMO10C from Streptomyces coelicolor We observed that the linker is disordered and extended, creating distance between the CBM and the catalytic domain and allowing these domains to move independently of each other. Functional studies with cellulose nanofibrils revealed that most of the substrate-binding affinity of full-length ScLPMO10C resides in the CBM. Comparison of the catalytic performance of full-length ScLPMO10C and its isolated catalytic domain revealed that the CBM is beneficial for LPMO activity at lower substrate concentrations and promotes localized and repeated oxidation of the substrate. Taken together, these results provide a mechanistic basis for understanding the interplay between catalytic domains linked to CBMs in LPMOs and CAZymes in general.
Highlights
Lytic polysaccharide monooxygenases (LPMOs) are copperdependentenzymesthatcatalyzetheoxidativecleavageofpolysaccharides such as cellulose and chitin, a feature that makes them key tools in industrial biomass conversion processes
These results provide a mechanistic basis for understanding the interplay between catalytic domains linked to carbohydrate-binding modules (CBMs) in LPMOs and carbohydrate-active enzymes (CAZymes) in general
The average radius of gyration for the model ensemble calculated by YASARA [32] was 40 Ϯ 10 Å, and the z-average hydrodynamic radius determined by dynamic light scattering was 35 Ϯ 9.5 Å (Fig. S2); a hydrodynamic radius of about 25 Å would be expected for a spherical protein of similar mass
Summary
Like many other industrially important cellulose-degrading enzymes, a considerable fraction of LPMOs contain a carbohydrate-binding module (CBM) Such CBMs may be connected to the catalytic domain through a variety of linkers, differing in length, sequence, and flexibility [20]. These linkers often contain regions of low sequence complexity and are predicted to be extended and flexible The nature of these linkers hampers structural studies of the full-length proteins, such studies could yield important insights into the interplay between the domains, substrate binding, and overall enzyme functionality. We have solved the first NMR structure of an LPMO-associated CBM (ScCBM2) and generated a structural model for fulllength ScLPMO10C by combining the structures of ScCBM2 and ScAA10 with the dihedral angle constraints derived from the secondary chemical shift data for the linker region. The 221–269 region, including the linker, 235–264 region, is indicated by a box
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