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

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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.

About the authors

A. A Pometun

Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences;Faculty of Chemistry, Lomonosov Moscow State University

Email: aapometun@gmail.com
119071 Moscow, Russia;119991 Moscow, Russia

L. A Shaposhnikov

Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences;Faculty of Chemistry, Lomonosov Moscow State University

119071 Moscow, Russia;119991 Moscow, Russia

S. A Zubanova

Faculty of Chemistry, Lomonosov Moscow State University

119991 Moscow, Russia

R. P Kovalevskii

Faculty of Chemistry, Lomonosov Moscow State University

119991 Moscow, Russia

D. L Atroshenko

Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences;Faculty of Chemistry, Lomonosov Moscow State University

119071 Moscow, Russia;119991 Moscow, Russia

E. V Pometun

Sechenov First Moscow State Medical University

119991 Moscow, Russia

S. S Savin

Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences;Faculty of Chemistry, Lomonosov Moscow State University

119071 Moscow, Russia;119991 Moscow, Russia

V. I Tishkov

Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences;Faculty of Chemistry, Lomonosov Moscow State University

Email: vitishkov@gmail.com
119071 Moscow, Russia;119991 Moscow, Russia

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