Abstract
BackgroundThe discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10).ResultsHere, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation.ConclusionsTogether, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.
Highlights
The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation
Seminal studies over the last decade have revealed the existence of copper-dependent lytic polysaccharide monooxygenases (LPMOs), which oxidatively cleave the cellulose chain thereby potentiating the activity of hydrolytic enzymes [23,24,25,26,27,28,29]
Primary sequence analysis of Cellulomonas catalytic modules All Cellulomonas LPMOs are multi-modular proteins containing a SEC pathway secretion signal peptide, a catalytic Activity Family 10 (AA10) module, and a C-terminal CBM2 that is indicative of cellulose or chitin binding [45, 69,70,71]
Summary
The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Seminal studies over the last decade have revealed the existence of copper-dependent lytic polysaccharide monooxygenases (LPMOs), which oxidatively cleave the cellulose chain thereby potentiating the activity of hydrolytic enzymes (chitin-, starch-, and hemicellulose-active LPMOs are known) [23,24,25,26,27,28,29]. In addition to boosting the activity of hydrolytic enzymes for nutrient acquisition, LPMOs have recently been implicated in other biological roles, including plant defense [36] and microbial copper acquisition and virulence [37]
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