Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

Lytic polysaccharidemonooxygenases (LPMOs) are surface-activeredox enzymes that catalyze the degradation of recalcitrant polysaccharides,making them important tools for energy production from renewable sources.In addition, LPMOs are important virulence factors for fungi, bacteria,and viruses. However, many knowledge gaps still exist regarding theircatalytic mechanism and interaction with their insoluble, crystallinesubstrates. Moreover, conventional structural biology techniques,such as X-ray crystallography, usually do not reveal the protonationstate of catalytically important residues. In contrast, neutron crystallographyis highly suited to obtain this information, albeit with significantsample volume requirements and challenges associated with hydrogen'slarge incoherent scattering signal. We set out to demonstrate thefeasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibriocholerae as a target. GbpA is a multifunctional proteinthat is secreted by the bacteria to colonize and degrade chitin. Wedeveloped an efficient deuteration protocol, which yields >10 mgofpure 97% deuterated protein per liter expression media, which wasscaled up further at international facilities. The deuterated proteinretains its catalytic activity and structure, as demonstrated by small-angleX-ray and neutron scattering studies of full-length GbpA and X-raycrystal structures of its LPMO domain (to 1.1 & ANGS; resolution),setting the stage for neutron scattering experiments with its substratechitin.

Automated summary

Demonstrating the feasibility of neutron-based techniques for studying LPMOs proteins using N-acetylglucosamine-binding protein A (GbpA) as a target.