Amylase 2019; 3: 41–54 Research Article Sebastian J. Muderspach**, Tobias Tandrup**, Kristian E. H. Frandsen, Gianluca Santoni, Jens-Christian N. Poulsen, Leila Lo Leggio* Further structural studies of the lytic polysaccharide monooxygenase AoAA13 belonging to the starch-active AA13 family https://doi.org/10.1515/amylase-2019-0004 received July 12, 2019; accepted November 15, 2019. Abstract: Lytic polysaccharide monooxygenases (LPMOs) are recently discovered copper enzymes that cleave recalcitrant polysaccharides by oxidation. The structure of an Aspergillus oryzae LPMO from the starch degrading family AA13 (AoAA13) has previously been determined from an orthorhombic crystal grown in the presence of copper, which is photoreduced in the structure. Here we describe how crystals reliably grown in presence of Zn can be Cu-loaded post crystallization. A partly photoreduced structure was obtained by severely limiting the X-ray dose, showing that this LPMO is much more prone to photoreduction than others. A serial synchrotron crystallography structure was also obtained, showing that this technique may be promising for further studies, to reduce even further photoreduction. We additionally present a triclinic structure of AoAA13, which has less occluded ligand binding site than the orthorhombic one. The availability of the triclinic crystals prompted new ligand binding studies, which lead us to the conclusion that small starch analogues do not bind to AoAA13 to an appreciable extent. A number of disordered conformations of the metal binding histidine brace have been encountered in this and other studies, and we have previously hypothesized that this disorder may be a consequence of loss of copper. We performed molecular dynamics in the absence of active site metal, and showed that the dynamics in solution differ somewhat from the disorder observed in the crystal, though the extent is equally dramatic. Keywords: LPMO; starch degradation; photoreduction; substrate binding; molecular dynamics. Abbreviations AA, auxiliary activities; AnAA13, lytic polysaccharide monooxygenase from Aspergillus nidulans; AoAA13, lytic polysaccharide monooxygenase from Aspergillus oryzae; CAZy, Carbohydrate Active enZyme database; CBM, carbohydrate binding module; LPMO, lytic polysaccharide monooxygenase; MD, molecular dynamics; MoLPMO13A, lytic polysaccharide monooxygenase from Magnaporthe oryzae; NcAA13, lytic polysaccharide monooxygenase from Nerospora crassa; PDB, Protein Data Bank; PEG, polyethylene glycol; RMSD, root mean square deviation; SSX, serial synchrotron crystallography; TaAA9A, lytic polysaccharide monooxygenase from Thermoascus aurantiacus. 1 Introduction Lytic polysaccharide monooxygenases (LPMOs) are enzymes capable of boosting the degradation of recalcitrant polysaccharides, such as cellulose, chitin and starch (as reviewed in, e.g., [1-3]). They are metalloenzymes which, utilizing a single copper ion active-site in conjunction with external electron donors and molecular oxygen or hydrogen peroxide as co-substrates, have been observed to cleave the glycosidic bond oxidatively [2,4]. LPMOs are classified as auxiliary activities (AA) in the Carbohydrate Active enZymes database (CAZy) [5] and are to date differentiated into seven families: AA9, AA10, AA11, *Corresponding author: Leila Lo Leggio, Department of Chemistry, University of Copenhagen, 2100 København Ø, Denmark, E-mail: leila@chem.ku.dk Sebastian J. Muderspach, Tobias Tandrup, Kristian E. H. Frandsen, Jens-Christian N. Poulsen, Department of Chemistry, University of Copenhagen, 2100 København Ø, Denmark Gianluca Santoni, ESRF, Structural Biology Group, 71 avenue des Martyrs, 38027 Grenoble cedex, France **These authors contributed equally to the work Open Access. © 2019 Sebastian J. Muderspach et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution alone 4.0 License. Brought to you by | The Royal Library (Det Kongelige Bibliotek) - National Library of Denmark / Copenhagen University Library Authenticated Download Date | 1/15/20 1:24 PM