Learning from oligosaccharide soaks of crystals of an AA13 lytic polysaccharide monooxygenase: crystal packing, ligand binding and active‐site disorder

摘要

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper‐dependent enzymes discovered within the last ten years. They oxidatively cleave polysaccharides (chitin, lignocellulose, hemicellulose and starch‐derived), presumably making recalcitrant substrates accessible to glycoside hydrolases. Recently, the first crystal structure of an LPMO–substrate complex was reported, giving insights into the interaction of LPMOs with β‐linked substrates (Frandsen et al. , 2016). The LPMOs acting on α‐linked glycosidic bonds (family AA13) display binding surfaces that are quite different from those of LPMOs that act on β‐linked glycosidic bonds (families AA9–AA11), as revealed from the first determined structure (Lo Leggio et al. , 2015), and thus presumably the AA13s interact with their substrate in a distinct fashion. Here, several new structures of the same AA13 enzyme, Aspergillus oryzae AA13, are presented. Crystals obtained in the presence of high zinc‐ion concentrations were used, as they can be obtained more reproducibly than those used to refine the deposited copper‐containing structure. One structure with an ordered zinc‐bound active site was solved at 1.65?? resolution, and three structures from crystals soaked with maltooligosaccharides in solutions devoid of zinc ions were solved at resolutions of up to 1.10??. Despite similar unit‐cell parameters, small rearrangements in the crystal packing occur when the crystals are depleted of zinc ions, resulting in a more occluded substrate‐binding surface. In two of the three structures maltooligosaccharide ligands are bound, but not at the active site. Two of the structures presented show a His‐ligand conformation that is incompatible with metal‐ion binding. In one of these structures this conformation is the principal one (80% occupancy), giving a rare atomic resolution view of a substantially misfolded enzyme that is presumably rendered inactive.