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

Authors :
Iurchenko, T. S.
Loginova, A. A.
Sergeev, E. P.
Pometun, E. V.
Tishkov, V. I.
Savin, S. S.
Pometun, A. A.
Source :
Moscow University Chemistry Bulletin; Feb2023, Vol. 78 Issue 1, p42-53, 12p
Publication Year :
2023

Abstract

NAD<superscript>+</superscript>-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 ; the value of k<subscript>cat</subscript> has not changed. When T250H is replaced, an increase in is observed, k<subscript>cat</subscript> decreases from 20 to 13 s<superscript>–1</superscript>. The replacement of K368H led to a slight increase in , k<subscript>cat</subscript> decreased from 20 to 6 s<superscript>–1</superscript>. The introduction of TGA and AGA additional inserts in α-helix at the C-terminus of the enzyme led to an increase in and . A bigger effect was observed for —the difference was more than 10 times. For mutant SauFDH with insertions k<subscript>cat</subscript> significantly reduced to 4 s<superscript>–1</superscript>. 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. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
00271314
Volume :
78
Issue :
1
Database :
Complementary Index
Journal :
Moscow University Chemistry Bulletin
Publication Type :
Academic Journal
Accession number :
163293926
Full Text :
https://doi.org/10.3103/S0027131423010078