Obtaining Isotopically Labeled Preparations of Recombinant NAD+-Dependent Formate Dehydrogenase from Pathogen Staphylococcus aureus

Abstract

NAD⁺-dependent formate dehydrogenase from Staphylococcus aureus (SauFDH) plays a key role in the biofilm growth of this pathogen. Under these conditions, the reaction catalyzed by SauFDH is the main source of energy in the cell. Therefore, highly effective SauFDH inhibitors can be used as a basis for the development of fundamentally new drugs against S.aureus biofilms. The Nuclear Magnetic Resonance method is one of the most powerful and effective for studying the interaction of promising inhibitors with the enzyme globule. In this work, we optimized the cultivation conditions for the effective introduction of ²H, ¹³C and ¹⁵N isotopes into the enzyme, both individually and together. MALDI/TOF/TOF mass spectra of peptides of isotopically labeled enzyme preparations after tryptic hydrolysis of samples SauFDH ²H, SauFDH ²H,¹⁵N,¹²C and SauFDH ²H,¹⁵N,¹³C showed a uniform distribution of these isotopes in the protein molecule with a degree of enrichment of at least 95% in ²H and not less than 98% for ¹³C and ¹⁵N. This level of isotope enrichment is the maximum achievable in a research laboratory and is completely sufficient for NMR experiments.