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 PMO-2 from Neurospora crassa (NcPMO-2) was heterologously expressed in Pichia pastoris to facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressing NcPMO-2 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 NcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.
Department of Molecular and Structural Biochemistry, North Carolina State University, Campus Box 7622, Raleigh, NC 27695, USA.
William B
WB
O'Dell
Department of Molecular and Structural Biochemistry, North Carolina State University, Campus Box 7622, Raleigh, NC 27695, USA.
Paul D
PD
Swartz
Biology and Soft Matter Division, Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 37831, USA.
Kevin L
KL
Weiss
Department of Molecular and Structural Biochemistry, North Carolina State University, Campus Box 7622, Raleigh, NC 27695, USA.
Flora
F
Meilleur
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003461:Crystallography; D062610:Fungal Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009492:Neurospora crassa / Q000737:chemistry* / Q000201:enzymology; D033363:Neutron Diffraction; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072757:Protein Conformation, beta-Strand; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014961:X-Ray Diffraction
D062610:Fungal Polysaccharides; D005656:Fungal Proteins; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases
Pichia pastoris; glycoengineering; lytic polysaccharide monooxygenases; neutron protein crystallography