T. S. Iurchenko, A. A. Pometun, D. L. Atroshenko, T. A. Chubar, V. I. Polshakov, S. S. Savin, V. I. Tishkov
{"title":"Obtaining Isotopically Labeled Preparations of Recombinant NAD+-Dependent Formate Dehydrogenase from Pathogen Staphylococcus aureus","authors":"T. S. Iurchenko, A. A. Pometun, D. L. Atroshenko, T. A. Chubar, V. I. Polshakov, S. S. Savin, V. I. Tishkov","doi":"10.1134/S0003683824700066","DOIUrl":null,"url":null,"abstract":"<p>NAD<sup>+</sup>-dependent formate dehydrogenase from <i>Staphylococcus aureus</i> (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 <i>S.aureus</i> 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 <sup>2</sup>H, <sup>13</sup>C and <sup>15</sup>N 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 <sup>2</sup>H, SauFDH <sup>2</sup>H,<sup>15</sup>N,<sup>12</sup>C and SauFDH <sup>2</sup>H,<sup>15</sup>N,<sup>13</sup>C showed a uniform distribution of these isotopes in the protein molecule with a degree of enrichment of at least 95% in <sup>2</sup>H and not less than 98% for <sup>13</sup>C and <sup>15</sup>N. This level of isotope enrichment is the maximum achievable in a research laboratory and is completely sufficient for NMR experiments.</p>","PeriodicalId":466,"journal":{"name":"Applied Biochemistry and Microbiology","volume":"60 7","pages":"1475 - 1480"},"PeriodicalIF":1.0000,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Applied Biochemistry and Microbiology","FirstCategoryId":"99","ListUrlMain":"https://link.springer.com/article/10.1134/S0003683824700066","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
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.
期刊介绍:
Applied Biochemistry and Microbiology is an international peer reviewed journal that publishes original articles on biochemistry and microbiology that have or may have practical applications. The studies include: enzymes and mechanisms of enzymatic reactions, biosynthesis of low and high molecular physiologically active compounds; the studies of their structure and properties; biogenesis and pathways of their regulation; metabolism of producers of biologically active compounds, biocatalysis in organic synthesis, applied genetics of microorganisms, applied enzymology; protein and metabolic engineering, biochemical bases of phytoimmunity, applied aspects of biochemical and immunochemical analysis; biodegradation of xenobiotics; biosensors; biomedical research (without clinical studies). Along with experimental works, the journal publishes descriptions of novel research techniques and reviews on selected topics.