Abstract
NAD+-dependent formate dehydrogenase from thermotolerant yeast Ogataea parapolymorpha DL-1 (OpaFDH, EC 1.2.1.2) with the additional N-terminal Gly residue and its double mutant OpaFDH_AD were overexpressed in E. coli cells. The enzyme yield was, respectively, 6000 and 6200 units per liter of the cultivation medium. Purified enzymes were obtained as homogeneous preparations with a yield of 62%. The purification procedure included ultrasonic cell disruption, heat treatment of cell free extracts at 55°C for 15 min, and hydrophobic chromatography on the Phenyl Sepharose Fast Flow. Crystallization experiments with wild-type OpaFDH resulted in the preparation of crystals of the apo-form but not the holo-form. Crystals of the holo-form were prepared in the case of the OpaFDH_AD mutant in the presence of 7 mM NAD+ and 10 mM sodium azide. The size and quality of the crystals are sufficient to collect X-ray diffraction data and determine the enzyme three-dimensional structure.
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ACKNOWLEDGMENTS
The facility of the Industrial Biotechnology Center (Federal State Institution, Federal Research Centre “Fundamentals of Biotechnology,” Russian Academy of Sciences) was used in this study.
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This study was supported by the Russian Foundation for Basic Research (project no. 18-34-20098).
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А.А. Pometun, K.M. Boyko, and S. A. Zubanova contributed equally to this article.
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Translated by A. Boutanaev
Abbreviations: OpaFDH and CboFDH are, respectively, formate dehydrogenases from the Ogataea parapolymorpha DL-1 and Candida boidinii yeast; SauFDH and PseFDH are formate dehydrogenases from the bacteria of Staphylococcus aureus and Pseudomonas sp.101, respectively.
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Pometun, A.A., Boyko, K.M., Zubanova, S.A. et al. Preparation of Recombinant Formate Dehydrogenase from Thermotolerant Yeast Ogataea parapolymorpha and Crystallization of Its Apo- and Holo- Forms. Moscow Univ. Chem. Bull. 76, 49–55 (2021). https://doi.org/10.3103/S0027131421010120
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DOI: https://doi.org/10.3103/S0027131421010120