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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.
Moscow University Chemistry Bulletin.
2014, Vol. 55, No. 2, P. 98
   

Copyright (C) Chemistry Dept., Moscow State University, 2002
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