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A. A. Alekseeva, A. S. Petrov, V. V. Fedorchuk, E. A. Fedorchuk, T. A. Osipova, V. I. Tishkov
Change of formatedehydrogenaseisoelectric
point by rational design
Abstract
Four new
mutant forms of NAD+-dependent formatedehydrogenase (FDH, EC 1.2.1.2.) from bacterium Pseudomonas
sp 101 with substitutions Lys112Pro, Lys231Ala, Lys231Ser and Lys317Asn were
obtained to extend the pH optimum of stability by site-directed mutegenesis. The choice of the residues was based on amino
acid alignment of FDHs from different sources and
analysis of FDH three-dimentional structure. Kinetic
properties and thermal stability were studied for all obtained mutant forms. It
was revealed, that replacements in positions 112 and 231 lead to small
improvement of kinetic properties, but at the same time the mutant form
Lys317Asn showed the decrease of affinity with coenzyme. The study of thermal stability
showed, that substitutions in positions 112 and 231 caused slightly
destabilization effect, but replacement Lys317Asn caused the essential loss in
thermal stability. The isoelectric point decreased
for all mutant forms for about 0.1 unit.
Key words: formatedehydrogenase,
Pseudomonas sp.101, protein engineering, rational design, isoelectric
point.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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