Engineering the Active Site of Formate Dehydrogenase from Staphylococcus aureus: Introduction to the Structure of the Additional Loop and Histidine Residues

Iurchenko, T.S.; Loginova, A.A.; Sergeev, E.P.; Pometun, E.V.; Tishkov, V.I.; Savin, S.S.; Pometun, A.A.

Авторы: Iurchenko T.S., Loginova A.A., Sergeev E.P., Pometun E.V., Tishkov V.I., Savin S.S., Pometun A.A.
Журнал: Moscow University Chemistry Bulletin
Том: 78
Номер: 1
Год издания: 2023
Издательство: Allerton Press Inc.
Местоположение издательства: United States
Первая страница: 92
Последняя страница: 106
DOI: 10.3103/S0027131423010078
Аннотация: NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from pathogenic bacterium Staphylococcus aureus (SauFDH) differs significantly from other FDHs both in terms of primary structure and catalytic properties. A distinctive feature of SauFDH is the highest (about 2.5-3 times) specific activity compared to other formate dehydrogenases. At the same time, SauFDH has high Michaelis constants for both substrates. Based on the analysis of three-dimensional structures and the alignment of amino acid sequences, replacements promising in terms of changing catalytic parameters were selected. The replacement of I220H resulted in an increase in KMNAD+ ; the value of kcat has not changed. When T250H is replaced, an increase in KMNAD+ is observed, kcat decreases from 20 to 13 s-1 . The replacement of K368H led to a slight increase in KMNAD+, kcat decreased from 20 s-1 to 6 s-1 . The introduction of TGA and AGA additional inserts in α-helix at the C-terminus of the enzyme led to an increase in KMNAD+ and KMHCOO-. A bigger effect was observed for KMNAD+ - the difference was more than 10 times. For mutant SauFDH with insertions kcat significantly reduced to 4 s-1 . Similar results were observed for mutants with multipoint replacements. Thus, the C-terminal sequence has been shown to play an important role in the catalysis of SauFDH
Добавил в систему: Тишков Владимир Иванович

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Engineering the Active Site of Formate Dehydrogenase from Staphylococcus aureus: Introduction to the Structure of the Additional Loop and Histidine Residuesстатья

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