Study of the structure-function relationship of formate dehydrogenase– an important enzyme for Staphylococcus aureus biofilms by rational design

Iurchenko, T.S.; Bolotova, S.B.; Loginova, A.A.; Kargov, I.S.; Atroshenko, D.L.; Savin, S.S.; Pometun, E.V.; Tishkov, V.I.; Pometun, A.A.

Авторы: Iurchenko Tatiana S., Bolotova Seseg B., Loginova Anastasia A., Kargov Ivan S., Atroshenko Denis L., Savin Svyatoslav S., Pometun Evgenii V., Tishkov Vladimir I., Pometun Anastasia A.
Журнал: Biochimie
Том: 216
Год издания: 2024
Издательство: Elsevier BV
Местоположение издательства: Netherlands
Первая страница: 194
Последняя страница: 204
DOI: 10.1016/j.biochi.2023.10.016
Аннотация: NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from the bacterium Staphylococcus aureus (SauFDH) plays an important role in the vital activity of this bacterium, especially in the form of biofilms. Understanding its mechanism and structure-function relationship can help to find special inhibitors of this enzyme, which can be used as medicines against staphylococci. The gene encoding SauFDH was successfully cloned and expressed in our laboratory. This enzyme has the highest kcat value among the described FDHs and also has a high temperature stability compared to other enzymes of this group. That is why it can also be considered as a promising catalyst for NAD(P)H regeneration in the processes of chiral synthesis with oxidoreductases. In this work, the principle of rational design was used to improve SauFDH catalytic efficiency. After bioinformatics analysis of the amino acid sequence in combination with visualization of the enzyme structure (PDB 6TTB), 9 probable catalytically significant positions 119, 194, 196, 217-219, 246, 303 and 323 were identified, and 16 new mutant forms of SauFDH were obtained and characterized. The introduction of the mentioned substitutions in most cases leads to a decrease in stability at high temperatures and an increase at low temperatures. Substitutions in positions 119 and 194 lead to a decreasing of KMNAD+. A consistent decrease in the Michaelis constant in the Ile-Val-Ala-Gly series at position 119 of SauFDH is shown. KMNAD+ of mutant SauFDH V119G decreased by 27 times compared to the wild-type enzyme. After substitution Phe194Val KMNAD+ decreased by 3.5 times. The catalytic constant for this mutant form practically did not change. For this mutant form, an increase in catalytic efficiency was demonstrated through the use of a multicomponent buffer system.
Добавил в систему: Тишков Владимир Иванович

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Study of the structure-function relationship of formate dehydrogenase– an important enzyme for Staphylococcus aureus biofilms by rational designстатья

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