Multi point mutagenesis of formate dehydrogenase

Galanicheva, N.; Parshin, P.; Shaposhnikov, L.; Atroshenko, D.; Savin, S.; Pometun, A.; Tishkov, V.

Авторы: Galanicheva N., Parshin P., Shaposhnikov L., Atroshenko D., Savin S., Pometun A., Tishkov V.
Журнал: Protein Science
Том: 30
Номер: S1
Год издания: 2021
Издательство: Cold Spring Harbor Laboratory Press
Местоположение издательства: United States
Первая страница: 152
Последняя страница: 152
Аннотация: NAD(P)+-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) is a well-known enzyme for fundamental study and practical application. FDH from bacterium Pseudomonas sp. 101 (PseFDH) is one of the most stable enzyme among the known FDHs. Formate dehydrogenase is actively used for coenzyme regeneration of NADH or NADPH in different biocatalytic processes as well as for determination of formate and NAD+ in complex samples.Previously, single point mutant PseFDHs (S131A, E170D, S160A) as well as multi point mutant PseFDH SM4S (major amino acid changes C145S, A198G and C255A) with increased thermal and chemical stability and/or improved Km for both substrates have been obtained in our laboratory. Previously, it was also found that mutation C145A in the wild-type enzyme resulted in improvement Km values followed by decrease of thermal stability. In this work positive amino acid changes S131A, E170D, S160A were sequentially introduced into PseFDH SM4S. In final mutant PseFDH Ser145 was changed with Ala residue.Analysis of thermal inactivation kinetics revealed, that the most thermal stability mutant form is PseFDH SM4S S131A/E170D/S160A. The substitution of glutamic for aspartic acid at position 170, together with the substitution of serine for alanine at positions 131, 160 leads to significant stabilization of PseFDH. The other PseFDH mutants showed stability similar to PseFDH SM4S. The mutant form SM4S/S131A/E170D/S160A/S145A has the highest catalytic efficiency for both substrates.This work was financed by the grant of the President of the Russian Federation to support young Russian scientists (MD- 349.2021.1.4).
Добавил в систему: Тишков Владимир Иванович

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