NAD<sup>+</sup>-dependent format dehydrogenase from the thermotolerant yeast <i>ogataea parapolymorpha</i>: properties and protein engineering of the <i>n</i>-terminal sequence

Abstract

Previously, the gene of formate dehydrogenase (FDH, EC 1.2.1.2) from the thermotolerant methylotrophic yeast Ogataea parapolymorpha DL 1 (OpaFDH) was cloned in our laboratory. The recombinant enzyme with an additional glycine amino acid residue (OpaFDH_GK) was obtained in Escherichia coli cells in an active and soluble form with a yield of more than 1 g per liter of medium. In the present work, a detailed comparison of this enzyme with FDH from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate the effect of the N-terminal residue on the properties of the enzyme, OpaFDH_K (identical to natural) and OpaFDH_AK variants containing an additional Ala residue at the N-terminus were also obtained. It was shown that the addition of an Ala residue to the N-terminus reduces the rate constant of thermal inactivation four times compared with the addition of a Gly residue. The addition of six more histidine residues to the N-terminus of OpaFDH_AK leads to an acceleration of purification, practically does not affect the kinetic parameters, but somewhat reduces the temperature stability, which, however, can be restored to the level of OpaFDH_AK by adding 0.5 M NaCl.