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

Pometun, A.A.; Boyko, K.M.; Yurchenko, T.S.; Nikolaeva, A.Y.; Atroshenko, D.L.; Savin, S.S.; Popov, V.O.; Tishkov, V.I.

Авторы: Pometun A.A., Boyko K.M., Yurchenko T.S., Nikolaeva A.Yu, Atroshenko D.L., Savin S.S., Popov V.O., Tishkov V.I.
Журнал: Biochemistry (Moscow)
Том: 85
Номер: 6
Год издания: 2020
Издательство: Pleiades Publishing, Ltd
Местоположение издательства: Road Town, United Kingdom
Первая страница: 689
Последняя страница: 696
DOI: 10.1134/S0006297920060061
Аннотация: 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. Resolving the 3D structure of the enzyme might be helpful in the search for highly specific SauFDH inhibitors that can be used as antibacterial agents against S. aureus biofilms. Here, we produced 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 selected 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. The apo- and holoforms of SauFDH had different crystal space groups, which suggest coenzyme binding by the SauFDH holoenzyme
Добавил в систему: Тишков Владимир Иванович

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

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