The Linker Region Promotes Activity and Binding Efficiency of Modular LPMO towards Polymeric Substrate

Abstract

ABSTRACT Lytic polysaccharide monooxygenases (LPMOs) mediate oxidative degradation of plant polysaccharides. The genes encoding LPMOs are most commonly arranged with one catalytic domain, while a few are found tethered to additional noncatalytic units, i.e., cellulase linker and carbohydrate-binding module (CBM). The presence of CBM is known to facilitate catalysis by directing the enzymes toward cellulosic polymer, while the role of linkers is poorly understood. Based on limited experimental evidence, linkers are believed to serve merely as flexible spacers between the structured domains. Thus, this study aims to unravel the role of the linker regions present in LPMO sequences. For this, we analyzed the genome of Botrytis cinerea and found 9 genes encoding cellulose lytic monooxygenases (AA9 family), of which BcAA9C was overexpressed in cellulose-inducible conditions. We designed variants of flLPMO (full-length enzyme) with truncation of either linker or CBM to examine the role of linker in activity, binding, and thermal stability of the associated monooxygenase. Biochemical assays predicted that the deletion of linker does not impact the potential of flLPMO for catalyzing the oxidation of Amplex Red, but that it does have a major influence on the capability of flLPMO to degrade recalcitrant polysaccharide substrate. Langmuir isotherm and SEM analysis demonstrated that linker domain aids in polysaccharide binding during flLPMO-mediated deconstruction of plant cell wall. Interestingly, linker domain was also found to contribute toward the thermostability of flLPMO. Overall, our study reveals that linker is not merely a spacer, but plays a key role in LPMO-mediated biomass fibrillation; these findings are broadly applicable to other polysaccharide-degrading enzymes. IMPORTANCE The polysaccharide-disintegrating carbohydrate-active enzymes (CAZymes) are often found with multimodular architecture, where the catalytic domain is connected to an accessory CBM domain with the help of a flexible linker region. So far, the linker has been understood merely as a flexible spacer between the two domains. Therefore, the current study is designed to determine the role of linker in polysaccharide fibrillation. To conceive this study, we have selected LPMO as a model enzyme, as it is not only an industrially relevant enzyme but it also harbors a catalytic domain, linker region, and CBM domain. The present study highlighted the crucial and indispensable role of the linker region in mediating polysaccharide disintegration. Considering its role in binding, thermostability, and activity toward polysaccharide substrate, we propose linker as a potential candidate for future CAZyme engineering.

Keywords

• linker
• carbohydrate-binding module
• CAZymes
• cellulose
• B. cinerea