2Chemistry Department, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-2742
3Innovations and High Technologies MSU Ltd., ul. Tsimlyanskaya 16, office 96, 109559 Moscow, Russia; fax: (495) 939-3208
4POMALEX Ltd, Kooperativnaya ul. 2, 140180 Zhukovsky, Moscow Region, Russia
5Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, 119334 Moscow, Russia; fax: (499) 135-8012
* To whom correspondence should be addressed.
Received July 3, 2012; Revision received July 9, 2012
Recombinant formate dehydrogenase (FDH, EC 22.214.171.124) 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 (Tm) 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 Tm 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.
KEY WORDS: formate dehydrogenase, thermal stability, rational design, catalytic efficiency