NAD+-Dependent Formate Dehydrogenase from Themotolerant Yeast Ogataea parapolymorpha: Properties and Protein Engineering of the N-Terminal Sequence

• Published in: Biochemistry (Moscow) Vol. 88; no. 9; pp. 1378 - 1389
• Main Authors: Pometun, Anastasia A., Shaposhnikov, Leonid A., Zubanova, Sofiya A., Kovalevskii, Rostislav P., Atroshenko, Denis L., Pometun, Evgenii V., Savin, Svyatoslav S., Tishkov, Vladimir I.
• Format: Journal Article
• Language: English
• Published: Moscow Pleiades Publishing 01-09-2023; Springer Nature B.V
• Subjects: Amino acids; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Dehydrogenase; Dehydrogenases; E coli; Enzymes; Formate dehydrogenase; Glycine; Histidine; Inactivation; Life Sciences; Microbiology; N-Terminus; Protein engineering; Residues; Sodium chloride; Thermal stability; Yeast; Yeasts; site-directed mutagenesis; catalytic properties; expression in; thermotolerant yeast; purification; formate dehydrogenase; DL 1; thermal stability
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297923090171

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. Recombinant enzyme with additional glycine amino acid residue (OpaFDH_GK) was obtained in
Escherichia coli
cells in active and soluble form with a yield of more than 1 g per liter of the medium. In the present work, a detailed comparison of this enzyme with FDHs from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate effect of 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 addition of an Ala residue to the N-terminus reduces four-fold the rate constant of thermal inactivation compared with the addition of a Gly residue. Addition of six more histidine residues to the N-terminus of OpaFDH_AK leads to acceleration of purification, practically does not affect kinetic parameters, but somewhat reduces thermal stability, which, however, can be restored to the level of OpaFDH_AK stability by adding 0.5 M NaCl.