期刊
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS
卷 75, 期 -, 页码 159-165出版社
INT UNION CRYSTALLOGRAPHY
DOI: 10.1107/S2053230X19000190
关键词
soluble fumarate reductase; anaerobiosis; Osm1; limited proteolysis; stabilization; crystallization
资金
- Chung-Ang University
- Basic Science Research Program through the National Research Foundation of Korea (NRF) of the Ministry of Education, Science and Technology [NRF-2014R1A1A1003451]
Osm1, a soluble fumarate reductase from Saccharomyces cerevisiae, is localized in both the mitochondria and the endoplasmic reticulum (ER). OSM1 genetically interacts with ERO1, which encodes an essential ER oxidoreductase for disulfide-bond formation under anaerobic conditions. However, the detailed enzymatic mechanisms involved in this interaction and the cellular roles of Osm1 are not fully understood. In this study, monomeric and stable recombinant Osm1 was successfully prepared for structural study. During purification, it was realized that the majority of recombinant Osm1 expressed in Escherichia coli lacked the flavin adenosine dinucleotide (FAD) cofactor. However, exogenously introduced FAD could be incorporated into recombinant Osm1, generating stable and homogenous holo Osm1. Moreover, after removing a flexible fragment by limited proteolysis, holo Osm1 formed isotropic crystals that retained catalytic activity. X-ray diffraction data were successfully collected from the Osm1 crystals to a resolution of 1.75 angstrom.
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