Frandsen Kristian E H, Poulsen Jens Christian Navarro, Tovborg Morten, Johansen Katja S, Lo Leggio Leila
Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.
Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark.
Acta Crystallogr D Struct Biol. 2017 Jan 1;73(Pt 1):64-76. doi: 10.1107/S2059798316019641.
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 Å resolution, and three structures from crystals soaked with maltooligosaccharides in solutions devoid of zinc ions were solved at resolutions of up to 1.10 Å. 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.
裂解多糖单加氧酶（LPMOs）是一类在过去十年中发现的铜依赖性酶。它们氧化裂解多糖（几丁质、木质纤维素、半纤维素和淀粉衍生物），推测使顽固的底物能够被糖苷水解酶作用。最近，报道了LPMO-底物复合物的首个晶体结构，这为了解LPMOs与β-连接底物的相互作用提供了线索（弗兰森等人，2016年）。作用于α-连接糖苷键的LPMOs（AA13家族）显示出与作用于β-连接糖苷键的LPMOs（AA9-AA11家族）截然不同的结合表面，正如首个确定的结构所揭示的那样（洛·莱焦等人，2015年），因此推测AA13s以独特的方式与它们的底物相互作用。在此，展示了同一AA13酶米曲霉AA13的几个新结构。使用在高锌离子浓度下获得的晶体，因为它们比用于优化已沉积的含铜结构的晶体更可重复获得。一个具有有序锌结合活性位点的结构在1.65 Å分辨率下解析，以及三个来自在无锌离子溶液中用麦芽寡糖浸泡的晶体的结构在高达1.10 Å的分辨率下解析。尽管晶胞参数相似，但当晶体耗尽锌离子时，晶体堆积会发生小的重排，导致底物结合表面更加封闭。在三个结构中的两个中，麦芽寡糖配体被结合，但不在活性位点。所展示的两个结构显示出一种与金属离子结合不相容的组氨酸-配体构象。在这些结构中的一个中，这种构象是主要构象（占有率80%），给出了一个罕见的原子分辨率视图，即一个可能无活性的严重错误折叠的酶。
