Tangled up in fibers: How a lytic polysaccharide monooxygenase binds its chitin substrate

Lytic polysaccharide monooxygenases (LPMOs) are redox enzymes that bind to and oxidize insoluble carbohydrate substrates, such as chitin or cellulose. This class of enzymes has attracted considerable attention due to their ability to convert biomaterials of high abundance into oligosaccharides that can be useful for producing biofuels and bioplastics. However, processes at the interface between solution and insoluble substrates represent a major challenge to biochemical and structural characterization. Here, we investigated the four-domain LPMO from Vibrio cholerae, N-acetyl glucosamine binding protein A (GbpA), to elucidate how it docks onto its insoluble substrate with its two terminal domains. First, we developed a protocol that allowed GbpA and chitin to form a stable complex in suspension, overcoming incompatibilities of the two binding partners with respect to pH. Using contrast variation small-angle neutron scattering (SANS), after determining the neutron scattering contrast match point for chitin (47% D2O), we characterized the structure of GbpA in complex with chitin by SANS, and by electron microscopy. We found that GbpA binds rapidly to chitin, where it spreads out on the chitin fibers, and smoothens their surface. In some locations, GbpA binding induces the formation of protein-chitin clumps containing hundreds of GbpA molecules. Together, this suggests how the secretion of GbpA efficiently prepares the ground for microcolony formation by the bacteria.