Multipoint Precision Binding of Substrate Protects Lytic Polysaccharide Monooxygenases from Self-Destructive...

Area of Science:

• Biochemistry
• Enzymology
• Biotechnology

Background:

• Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes critical for biomass degradation.
• Their structural features governing functionality, particularly substrate interaction, remain poorly understood.
• Understanding LPMO structure-function relationships is vital for industrial applications.

Purpose of the Study:

• To investigate the role of conserved amino acids on the substrate-binding surface of a chitin-active LPMO (CBP21/SmLPMO10A).
• To elucidate how substrate binding influences LPMO stability and catalytic activity.
• To identify mechanisms of LPMO inactivation.

Main Methods:

• Site-directed mutagenesis of 13 conserved residues on the CBP21 substrate-binding surface.
• Enzyme activity assays and product formation analysis over time.
• Proteomics to assess protein integrity and identify oxidation sites.

Main Results:

• Mutations affecting substrate binding, not the catalytic site, significantly impacted enzyme performance and stability.
• Loss of activity was linked to oxidation of active site residues.
• Reduced enzyme stability correlated with decreased chitin binding, indicating substrate adhesion protects against inactivation.

Conclusions:

• LPMOs possess extended, adaptable surfaces for multipoint substrate binding, crucial for enzyme stability.
• Substrate binding provides confinement, enabling control over LPMO's potent oxidative activity.
• Findings inform optimized industrial use and design of LPMO-inspired catalysts.