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

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Resumo

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.

Sobre autores

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. 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. Zubanova

Faculty of Chemistry, Lomonosov Moscow State University

119991 Moscow, Russia

R. Kovalevskii

Faculty of Chemistry, Lomonosov Moscow State University

119991 Moscow, Russia

D. 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. Pometun

Sechenov First Moscow State Medical University

119991 Moscow, Russia

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. 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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