Highly Stable Mutant Bacterial Formate Dehydrogenase with Improved Catalytic Properties

Pometuna, A.A.; Shirokova, A.A.; Galanicheva, N.P.; Shaposhnikov, L.A.; Atroshenko, D.L.; Pometun, E.V.; Tishkov, V.I.; Savin, S.S.

Авторы: Pometuna A.A., Shirokova A.A., Galanicheva N.P., Shaposhnikov L.A., Atroshenko D.L., Pometun E.V., Tishkov V.I., Savin S.S.
Журнал: Moscow University Chemistry Bulletin
Том: 78
Номер: 1
Год издания: 2023
Издательство: Allerton Press Inc.
Местоположение издательства: United States
Первая страница: 20
Последняя страница: 27
DOI: 10.3103/S0027131423010042
Аннотация: NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from methylotrophic bacteriumPseudomonas sp.101 (PseFDH) has one of the highest thermal stability among all known enzymes of thisgroup. The introduction of a number of amino acid substitutions into PseFDH made it possible to obtain a multipoint mutant PseFDH SM4S enzyme with even higher temperature and chemical stability. Previously, we showed that the introduction of additional single point replacements S131A, or S160A, or E170D into PseFDH SM4S led to further stabilization of the enzyme. In this work, based on the PseFDH SM4S S131A mutant, new mutant FDHs obtained, in which, compared to PseFDH SM4S, we added double S131A/E170D (M2), triple S131A/S160A/E170D (M3) and quadruple S131A/S160A/E170D/S145A (PseFDH SM4A M3)amino acids replacements. The new PseFDH mutants were overexpressed in E. coli cells, purified and characterized. The S131A/E170D and S131A/S160A/E170D changes provided further improving thermal stability.The introduction of the S145A substitution into PseFDH SM4A M3 leads to a significant decrease in and while maintaining the catalytic constant at the same level. This mutant form can be successfully used in NADH regeneration systems, as well as for the detection of NAD+ and formate in biological systems.
Добавил в систему: Тишков Владимир Иванович

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Highly Stable Mutant Bacterial Formate Dehydrogenase with Improved Catalytic Propertiesстатья

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