The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation

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 LPMO10C from 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 LPMO10C resides in the CBM. Comparison of the catalytic performance of full-lengthLPMO10C 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.

Influence of Lytic Polysaccharide Monooxygenase Active Site Segments on Activity and Affinity

“…Since NcLPMO9C ∆CBM is lacking the CBM1 it is conceivable that the latter domain helps the enzyme to stay close to the substrate and therefore promotes the formation of even smaller products. This effect has been described by Courtade et al [26]. For the conversion of XG, the presence of the CBM1 is not necessary since similar conversion rates of NcLPMO9C and NcLPMO9C ∆CBM were observed.…”

“…In contrast to Chalak and coworkers [32] no change in the regioselectivity of the enzyme was observed when removing the CBM1. However, recent efforts to elucidate the role of a family 2 CBM linked to LPMO10C from Streptomyces coelicolor revealed no change in regioselectivity upon truncation of the CBM2 from the enzyme [26]. Furthermore, the of 22 same study also showed that the quantity of formed products was decreased.…”

“…Interestingly, removal of the CBM1 from the C1/C4 oxidizing Podospora anserina LPMO9H, not only negatively affected binding and activity, but also resulted in a shift in regioselectivity towards predominantly C1 oxidation [32]. Similarly, other studies reported that truncation of the linker and CBM1 of LPMOs affect both the substrate binding and the formed products [24,26].…”

“…Regioselectivity defines whether LPMOs oxidize glucosyl residues in cellulose at the C1 or C4 position, or both [19]. Studies to link sequences and structural features to the regioselectivity of LPMO have been attempted [19][20][21][22][23][24][25][26][27][28][29][30]. So far, although similarities have occurred, such phylogenetic analysis could not unambiguously predict the three LPMO regioselectivities.…”

Current understanding of substrate specificity and regioselectivity of LPMOs

“…Domain similarity network analysis has shown the correlation between the additional domains and the substrate specificity of the full enzymes (Book et al 2014;Zhou et al 2019b). CBM truncation studies have been reported for both LPMO9s and LPMO10s (Chalak et al 2019;Courtade et al 2018;Crouch et al 2016;Forsberg et al 2016;Laurent et al 2019). Comparison of the performance of LPMOs with and without CBMs have shown that, deletion of CBMs reduced LPMO's binding capacity to crystalline substrates, especially at low substrate concentrations.…”