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Odintseva Ye. R., Popova A. S., Rozhkova A. M., Tishkov V. I.
Role of the Cysteine Remains in Stability of Bacterial Formiate
Dehydrogenase
Abstract
Bacterial NAD+ depended formate dehydrogenase (FDH; EC
1.2.1.2) from Pseudomonas sp.101 (PseFDH) is the most stable enzyme
among all FDHs known. Inactivation of the PseFDH below 45°C occurs due to
oxidation of SH-groups of the enzyme. According to experiments for modification
FDH by specific reagents there are two Cys residues essential for FDH catalytic
activity. Analysis of three-dimensional FDH structure shows that residues
Cys255 and Cys354 are located on the surface of globule. As was shown earlier,
the replacement of Cys255 by Ser and Met resulted in increase of enzyme
stability at 25°C more then 100
fold and decrease of thermal stability (above 45°C) by 3 and 10 fold,
respectively, and decrease of the enzyme affinity for NAD+. Mutant
FDH Cys255Ala prepared in this work is stable as Cys255Ser with saving the
kinetic parameters of native enzyme. Input of mutations increasing the thermal
stability of FDH in temperature above 45°C resulted in multi-point
mutant FDH with the same thermal stability as the wild-type enzyme. The mutant
FDHs Cys354Ala, Cys354Ser and Cys354Met were also obtained in this work and
their properties were studied.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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