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Anastasia A. Pometun, Anna A. Shirokova, Natalia P. Galanicheva, Leonid A. Shaposhnikov, Denis L. Atroshenko, Evgenii V. Pometun, Vladimir I. Tishkov, Svyatoslav S. Savin
Highly
stable mutant bacterial format dehydrogenase with improved catalytic properties
Abstract
Abstract. NAD+-dependent
formate dehydrogenase (FDH, EC 1.2.1.2) from methylotrophic bacterium Pseudomonas
sp.101 (PseFDH) has one of the highest thermal stability among all known
enzymes of this group. The introduction of a number of amino acid substitutions
into PseFDH made it possible to obtain a multipoint mutant PseFDH SM4S enzyme with even higher temperature and chemical stability.
Previously, we showed that the introduction of additional single point
replacements S131A, or S160A, or E170D into PseFDH SM4S led to further
stabilization of the enzyme. In this work, based on the PseFDH SM4S S131A
mutant, new mutant FDHs obtained, in which, compared to PseFDH SM4S, we added
double S131A/E170D (M2), triple S131A/S160A/E170D (M3) and quadruple
S131A/S160A/ E170D/S145A (PseFDH SM4A M3) amino acids replacements. The new
PseFDH mutants were overexpressed in E. coli cells, purified and
characterized. The S131A/E170D and S131A/S160A/E170D changes provided further
improving thermal stability. The introduction of the S145A substitution into
PseFDH SM4S M4 leads to a significant decrease in KMNAD+
and KMHCOO– while maintaining the catalytic
constant at the same level. This mutant form can be successfully used in NADH
regeneration systems, as well as for the detection of
NAD+ and formate in biological systems.
Key words: formate dehydrogenase, Pseudomonas sp.101, catalytic properties,
thermal stability, site-directed mutagenesis
Copyright (C) Chemistry Dept., Moscow State University, 2002
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