title: Stabilization of plant formate dehydrogenase by rational design.
abstract: Recombinant formate dehydrogenase (FDH, EC 1.2.1.2) from soy Glycine max (SoyFDH) has the lowest values of Michaelis constants for formate and NAD+ among all studied formate dehydrogenases from different sources. Nevertheless, it also has the lower thermal stability compared to enzymes from bacteria and yeasts. The alignment of full sequences of FDHs from different sources as well as structure of apo- and holo-forms of SoyFDH has been analyzed. Ten mutant forms of SoyFDH were obtained by site-directed mutagenesis. All of them were purified to homogeneity and their thermal stability and substrate specificity were studied. Thermal stability was investigated by studying the inactivation kinetics at different temperatures and by differential scanning calorimetry (DSC). As a result, single-point (Ala267Met) and double mutants (Ala267Met/Ile272Val) were found to be more stable than the wild-type enzyme at high temperatures. The stabilization effect depends on temperature, and at 52°C it was 3.6- and 11-fold, respectively. These mutants also showed higher melting temperatures in DSC experiments - the differences in maxima of the melting curves (T(m)) for the single and double mutants were 2.7 and 4.6°C, respectively. For mutations Leu24Asp and Val127Arg, the thermal stability at 52°C decreased 5- and 2.5-fold, respectively, and the T(m) decreased by 3.5 and 1.7°C, respectively. There were no differences in thermal stability of six mutant forms of SoyFDH - Gly18Ala, Lys23Thr, Lys109Pro, Asn247Glu, Val281Ile, and Ser354Pro. Analysis of kinetic data showed that for the enzymes with mutations Val127Arg and Ala267Met the catalytic efficiency increased 1.7- and 2.3-fold, respectively.
authors.affiliation: Bach Institute of Biochemistry, Russian Academy of Sciences, 117234 Moscow, Russia.
authors.forename: A A
authors.initials: AA
authors.lastname: Alekseeva
authors.forename: S S
authors.initials: SS
authors.lastname: Savin
authors.forename: S Yu
authors.initials: SY
authors.lastname: Kleimenov
authors.forename: I V
authors.initials: IV
authors.lastname: Uporov
authors.forename: E V
authors.initials: EV
authors.lastname: Pometun
authors.forename: V I
authors.initials: VI
authors.lastname: Tishkov
mesh_terms: D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D003198:Computer Simulation; D015195:Drug Design; D004795:Enzyme Stability; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D030262:Soybean Proteins; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics
chemical_list: D030262:Soybean Proteins; D005560:Formate Dehydrogenases