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首页> 外文期刊>Acta crystallographica. Section D, Structural biology >Structural and binding studies of a new chitin‐active AA10 lytic polysaccharide monooxygenase from the marine bacterium Vibrio campbellii
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Structural and binding studies of a new chitin‐active AA10 lytic polysaccharide monooxygenase from the marine bacterium Vibrio campbellii

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

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摘要

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 Kd of 0.1 ± 0.01?μM. In contrast, a Kd 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.

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