NAD.sup.+-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; p. 1378
• Main Authors: Pometun, Anastasia A, Shaposhnikov, Leonid A, Zubanova, Sofiya A, Kovalevskii, Rostislav P, Atroshenko, Denis L, Pometun, Evgenii V, Savin, Svyatoslav S
• Format: Journal Article
• Language: English
• Published: Springer 01-09-2023
• Subjects: Analysis; Identification and classification; Oxidoreductases; Properties; Protein engineering; Structure; Yeast fungi; Russia
• ISSN: 0006-2979
• 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.