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Tatiana S. Iurchenko, Anastasia A. Loginova, Egor P. Sergeev, Evgenii V. Pometun, Vladimir I. Tishkov, Svyatoslav S. Savin, Anastasia A. Pometun
Engineering the active site of formate dehydrogenase from Staphylococcus
aureus: introduction to the structure of the additional loop and histidine
residues
Abstract
Abstract. 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, substitutions 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 substitutions. Thus, the C-terminal sequence has been
shown to play an important role in the catalysis of SauFDH.
Key words: NAD+-dependent formate dehydrogenase, site-directed
mutagenesis, Staphylococcus aureus, structure, C-terminus,
modeling
Copyright (C) Chemistry Dept., Moscow State University, 2002
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