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

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• 作者：William B. O'Dell ; Paul D. Swartz ; Kevin L. Weiss and Flora Meilleur
• 关键词：lytic polysaccharide monooxygenases ; Pichia pastoris ; glycoengineering ; neutron protein crystallography
• 刊名：Acta Crystallographica Section F
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：73
• 期：2
• 页码：70-78
• 全文大小：1741K
• ISSN：1744-3091

文摘

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.