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A. A. Pometun, P. D. Parshin, N. P. Galanicheva, I. V. Uporov, D. L. Atroshenko, S. S. Savin, V. I. Tishkov
Influence of HIS6
sequence on properties of formate dehydrogenase from bacterium Pseudomonas
sp. 101
Abstract
NAD(P)+-dependent formate dehydrogenase
(FDH, EC 1.2.1.2.) is actively used in processes of chiral synthesis by
oxidoreductases with systems of reduced cofactor regeneration. Efficient use of
FDH in such systems requires simple and fast enzyme purification method.
Metal-chelating affinity chromatography is widely used for such purposes. The
method requires presence of six (or more) his residues at N- or C-terminus of
protein. Addition of extra His residues can influence enzyme properties.
Computer modeling of structure of FDH from bacterium Pseudomonas sp. 101
with different positions of His6 sequence showed that optimal case
is His-tag at N-terminus. Three types of PseFDH with His6 were
prepared – wild-type NAD+-dependent enzyme and two mutant NADP+-specific
forms. New PseFDHs were obtained as homogeneous preparations through one step
purification procedure. Comparison of properties of PseFDHs with and without
His-tag showed that they have similar kinetic properties.
Key words:
formate dehydrogenase, Pseudomonas sp.101, affinity chromatography,
catalytic properties, thermal stability, His-tag.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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