Structural and binding studies of a new chitin-active AA10 lytic polysaccharide monooxygenase from the marine bacterium Vibrio campbellii

Abstract

Vibrio spp. play a crucial role in the global recycling of the highly abundant recalcitrant biopolymer chitin in marine ecosystems through their ability to secrete chitin-degrading enzymes to efficiently hydrolyse chitinous materials and use them as their major carbon source. In this study, the first crystal structures of a complete four-domain chitin-active AA10 lytic polysaccharide monooxygenase from the chitinolytic bacterium Vibrio campbellii type strain ATCC BAA-1116 are reported. The crystal structures of apo and copper-bound VhLPMO10A were resolved as homodimers with four distinct domains: an N-terminal AA10 catalytic (CatD) domain connected to a GlcNAc-binding (GbpA_2) domain, followed by a module X domain and a C-terminal carbohydrate-binding module (CBM73). Size-exclusion chromatography and small-angle X-ray scattering analysis confirmed that VhLPMO10A exists as a monomer in solution. The active site of VhLPMO10A is located on the surface of the CatD domain, with three conserved residues (His1, His98 and Phe170) forming the copper(II)-binding site. Metal-binding studies using synchrotron X-ray absorption spectroscopy and X-ray fluorescence, together with electron paramagnetic resonance spectroscopy, gave consistently strong copper(II) signals in the protein samples, confirming that VhLPMO10A is a copper-dependent enzyme. ITC binding data showed that VhLPMO10A could bind various divalent cations but bound most strongly to copper(II) ions, with a K d of 0.1 ± 0.01 µM. In contrast, a K d of 1.9 nM was estimated for copper(I) ions from redox-potential measurements. The presence of ascorbic acid is essential for H2O2 production in the reaction catalysed by VhLPMO10A. MALDI-TOF MS identified VhLPMO10A as a C1-specific LPMO, generating oxidized chitooligosaccharide products with different degrees of polymerization (DP2ox–DP8ox). This new member of the chitin-active AA10 LPMOs could serve as a powerful biocatalyst in biofuel production from chitin biomass.

Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

“…Given the recently solved crystal structure of full-length VhLPMO10A fromV. campbellii, a close homologue of GbpA, NMX studies of FL-GbpA and homologues may also become a possibility . Neutron studies with SANS or NR can reveal how LPMOs interact with the carbohydrate substrates and how the LPMO structure adapts to carbohydrate surfaces or fibers.…”

“…While the structures of several LPMOs have been determined to atomic resolution by X-ray crystallography, including the first three domains of GbpA, and all four domains of the close homologue Vh LPMO10A from Vibrio campbellii, the functional information has been limited, in part due to the inability to map hydrogen atoms in the structures. For redox-active enzymes like LPMOs, neutron macromolecular crystallography (NMX) is a strong complementary method to X-ray crystallography that can reveal additional information .…”

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

“…The fourth domain (classified as a CBM73 in CAZy) also binds chitin [12], whereas domains 2 and 3, which structurally resemble flagellin and pilin-binding proteins, respectively, mediate attachment to the cell surface of the bacteria [12]. The structure of the first three domains of GbpA has been solved by X-ray crystallography [12], recently complemented with the full-length crystal structure of a GbpA homolog from Vibrio campbellii [14]. In addition, the solution structure of GbpA has been characterized with Small-Angle X-ray Scattering (SAXS) and Small-Angle X-ray Scattering (SANS) [12, 15], revealing a monomeric elongated structure.…”

Unravelling the secrets of multi-domain lytic polysaccharide monooxygenases (LPMOs)

“…In this respect, it is worth noting that the topology of the substrate-binding surfaces of the LPMO domains of GbpA and VhLPMO10A is somewhat different from those of LPMOs known to play a key role in chitin degradation, such as SmLPMO10A (also known as CBP21; see arrows in Fig. 3 in Zhou et al, 2023).…”

“…In this issue, Zhou et al present the crystal structure of a complete four-domain GbpA homologue (Zhou et al, 2023), VhLPMO10A, produced by Vibrio campbellii (also known as Vibrio harveyi), an abundant pathogen in marine ecosystems (Zhang et al, 2020). The previously published structure of GbpA (Wong et al, 2012) lacks the Cterminal CBM73, whereas the structure of CbpD (Dade et al, 2022) lacks both Module X (which is absent in this protein) and the CBM73.…”