The Role of Ala198 in the Stability and Coenzyme Specificity of Bacterial Formate Dehydrogenases

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Abstract


It has been shown by an X-ray structural analysis that the amino acid residues Ala198, which are located in the coenzyme-binding domain of NAD+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from bacteria Pseudomonas sp.101 and Moraxella sp. C-1 (PseFDH and MorFDH, respectively), have non-optimal values of the angles ψ and φ. These residues were replaced with Gly by site-directed mutagenesis. The mutants PseFDH A198G and MorFDH A198G were expressed in E.coli cells and obtained in active and soluble forms with more than 95% purity. The study of thermal inactivation kinetics showed that the mutation A198G results in a 2.5- fold increase in stability compared to one for the wild-type enzymes. Kinetic experiments indicate that A198G replacement reduces the K M NAD+ value from 60 to 35 and from 80 to 45 μM for PseFDH and MorFDH, respectively, while the K M HCOO- value remains practically unchanged. Amino acid replacement A198G was also added to the mutant PseFDH D221S with the coenzyme specificity changed from NAD + to NADP +. In this case, an increase in thermal stability was also observed, but the influence of the mutation on the kinetic parameters was opposite: KM increased from 190 to 280 μM and from 43 to 89 mM for NADP + and formate, respectively. According to the data obtained, inference could be drawn that earlier formate dehydrogenase from bacterium Pseudomonas sp. 101 was specific to NADP +, but not to NAD +.


A. A. Alekseeva

A.N. Bach Institute of Biochemistry; Innovations and High Technologies MSU Ltd

Author for correspondence.
Email: vitishkov@gmail.com

Russian Federation

V. V. Fedorchuk

Innovations and High Technologies MSU Ltd; M.V. Lomonosov Moscow State University

Email: vitishkov@gmail.com

Russian Federation

S. A. Zarubina

Innovations and High Technologies MSU Ltd; M.V. Lomonosov Moscow State University

Email: vitishkov@gmail.com

Russian Federation

E. G. Sadykhov

A.N. Bach Institute of Biochemistry

Email: vitishkov@gmail.com

Russian Federation

A. D. Matorin

M.V. Lomonosov Moscow State University

Email: vitishkov@gmail.com

Russian Federation

S. S. Savin

A.N. Bach Institute of Biochemistry; Innovations and High Technologies MSU Ltd; M.V. Lomonosov Moscow State University

Email: vitishkov@gmail.com

Russian Federation

V. I. Tishkov

A.N. Bach Institute of Biochemistry; Innovations and High Technologies MSU Ltd; M.V. Lomonosov Moscow State University

Email: vitishkov@gmail.com

Russian Federation

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Copyright (c) 2015 Alekseeva A.A., Fedorchuk V.V., Zarubina S.A., Sadykhov E.G., Matorin A.D., Savin S.S., Tishkov V.I.

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