Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction

Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate disrupting enzymes secreted by bacteria and fungi that break glycosidic bonds via an oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO₂ from Neurospora crassa (NcPMO₂) was heterologously expressed in Pichia pastoris to facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressing NcPMO₂ from a glycoengineered strain of P. pastoris and by the use of crystal seeding methods, respectively. These improvements resulted in high resolution (1.20 Å) X ray diffraction data collection at 100 K and the production of a large NcPMO₂ crystal suitable for room temperature neutron diffraction data collection to 2.12 Å resolution.