Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

H. V. Sørensen, Mateu Montserrat-Canals, Jennifer S.M. Loose, S. Zoë Fisher, Martine Moulin, Matthew P. Blakeley, Gabriele Cordara, Kaare Bjerregaard-Andersen, Ute Krengel

    Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskriftPeer review

    Sammanfattning

    Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox 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 their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen’s large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.

    Originalspråkengelska
    Sidor (från-till)29101-29112
    Antal sidor12
    TidskriftACS Omega
    Volym8
    Nummer32
    DOI
    StatusPublished - 2023 aug.

    Ämnesklassifikation (UKÄ)

    • Biokemi och molekylärbiologi
    • Strukturbiologi

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