2Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences, 119071 Moscow, Russia
3Innovations and High Technologies MSU Ltd., 109559 Moscow, Russia
4Kurchatov Institute National Research Center, 123182 Moscow, Russia
# These authors contributed equally to the work.
* To whom correspondence should be addressed.
Received April 16, 2020; Revised May 2, 2020; Accepted May 2, 2020
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.
KEY WORDS: formate dehydrogenase, Staphylococcus aureus, expression, purification, crystallization, X-ray diffraction analysis