|
Leonid A. Shaposhnikov, Svyatoslav S. Savin, Denis L. Atroshenko, Tatyana A. Chubar, Evgenii V. Pometun, Vladimir I. Tishkov, Anastasia A. Pometun
Engineering
the N-terminal sequence of Glycine max soybean formate dehydrogenase
Abstract
Abstract.NAD(P)+-dependent
formate dehydrogenase (FDH, EC 1.2.1.2.) catalyzes the oxidation of formate ion
with the coupled reduction of NAD(P)+ to NAD(P)H. Previously, in our
laboratory, a genetic construct was obtained with the soyfdh2 gene
encoding isoenzyme 2 of formate dehydrogenase from soybean Glycine max
(SoyFDH). In this construct the nucleotide sequence encoding the signal peptide
responsible for the transport of the pro-enzyme into the mitochondria of plant
cells (the
SoyFDH_L enzyme) was deleted. In this work, a second variant of SoyFDH_S was
obtained, in which, compared to SoyFDH_L, the sequence at the N-terminus was
reduced and changed to mimic the N-terminus sequence in FDH from Pseudomonas
sp.101 bacteria. Next, a sequence of six histidine residues (His-tag) was added to the
N-terminus of the long and short forms of SoyFDH. All four SoyFDH variants were
expressed in E. coli BL21(DE3)CodonPlus, these enzymes were purified,
their kinetic parameters were determined, and thermal stability was studied. In
the case of SoyFDH_L, which is similar to the natural variant of the enzyme,
both with and without His-tag, the expression level is two times higher
compared to the truncated variant. The addition of His-tag to the N-terminus of
enzymes reduces the level of expression. Changing the sequence of the
N-terminus, as well as introducing the His-tag sequence to the N-terminus, does
not significantly affect thermal stability of the enzymes at temperatures of
50–56 °C. However, due to the higher values of the activation enthalpy ΔH≠of the thermal
inactivation process, the shortened form at normal temperatures is 3 times more
stable than the natural one. A comparison of the kinetic parameters of the two
SoyFDH variants shows that the catalytic constants are the same, but the long
version of SoyFDH_L has lower values KMHCOO–, and the short version
has lower KMNAD+values. The introduction of
His-tag into the N-terminus of enzymes does not affect their kinetic
parameters.
Key words:formate dehydrogenase, Glycine
max, protein engineering, catalytic parameters, temperature stability
Copyright (C) Chemistry Dept., Moscow State University, 2002
|
|
