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

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Anastasia A. Pometun^1,2,a*, Leonid A. Shaposhnikov^1,2, Sofiya A. Zubanova², Rostislav P. Kovalevskii², Denis L. Atroshenko^1,2, Evgenii V. Pometun³, Svyatoslav S. Savin², and Vladimir I. Tishkov^1,2,b*

¹Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences, 119071 Moscow, Russia

²Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

³Sechenov First Moscow State Medical University, 119991 Moscow, Russia

^* To whom correspondence should be addressed.

Received June 12, 2023; Revised July 7, 2023; Accepted July 12, 2023

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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.

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KEY WORDS: formate dehydrogenase, Ogataea parapolymorpha DL 1, thermotolerant yeast, expression in E. coli, catalytic properties, purification, thermal stability, site-directed mutagenesis

DOI: 10.1134/S0006297923090171

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