Highly-Active Recombinant Formate Dehydrogenase from Pathogenic Bacterium Staphylococcus aureus: Preparation and Crystallization

Abstract

NAD+-dependent formate dehydrogenase from Staphylococcus aureus (SauFDH) is one of the key enzymes responsible for the survival of this pathogen in the form of biofilms. 3D structure of the enzyme might be helpful in the search for highly specific SauFDH inhibitors that can be used as antibacterial agents exactly against S. aureus biofilms. Here, we prepared a recombinant SauFDH in Escherichia coli cells with a yield of 1 g target protein per liter medium. The developed procedure for the enzyme purification allowed to obtain 400 mg of homogenous enzyme with 61% yield. The specific activity of the purified recombinant SauFDH was 20 U per mg protein, which was 2 times higher than the previously reported activities of formate dehydrogenases. We also found crystallization conditions in the course of two rounds of optimization and obtained 200- and 40-µm crystals for the SauFDH apo- and holoenzymes, respectively. X-ray analysis using synchrotron X-ray sources produced diffraction data sufficient for solving the three-dimensional structures of the apo- and holoenzymes with the resolution of 2.2 and 2.7 Å, respectively. Crystals of the apo- and holoforms of SauFDH had different crystal space groups, which suggest coenzyme binding in the SauFDH holoenzyme.

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Abbreviations

FDH:

formate dehydrogenase

SauFDH:

formate dehydrogenase from Staphylococcus aureus

PseFDH:

formate dehydrogenase from Pseudomonas sp. 101

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Acknowledgements

Some experiments were carried out using equipment from the Industrial Biotechnology Center of Collective Use, Fundamentals of Biotechnology Research Centre, Russian Academy of Sciences.

Funding

This work was supported by the Russian Foundation for Basic Research (projects Nos. 17-04-01662 and 20-04-00915, production and crystallization of enzymes), by the Federal Space Agency of Russia (Crystallizer experiment, crystallization and collection of X-ray diffraction data), and by the Ministry of Science and Higher Education of the Russian Federation (preliminary analysis of X-ray diffraction data).

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Correspondence to V. I. Tishkov.

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This article does not contain any studies with humans or animals performed by any of the authors. The authors declare no conflict of interest.

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Pometun, A., Boyko, K., Yurchenko, T. et al. Highly-Active Recombinant Formate Dehydrogenase from Pathogenic Bacterium Staphylococcus aureus: Preparation and Crystallization. Biochemistry Moscow 85, 689–696 (2020). https://doi.org/10.1134/S0006297920060061

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Keywords

  • formate dehydrogenase
  • Staphylococcus aureus
  • expression
  • purification
  • crystallization
  • X-ray diffraction analysis