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

Pometun, A.A.; Shaposhnikov, L.A.; Zubanova, S.A.; Kovalevskii, R.P.; Atroshenko, D.L.; Pometun, E.V.; Savin, S.S.; Tishkov, V.I.

Авторы: Pometun Anastasia A., Shaposhnikov Leonid A., Zubanova Sofiya A., Kovalevskii Rostislav P., Atroshenko Denis L., Pometun Evgenii V., Savin Svyatoslav S., Tishkov Vladimir I.
Журнал: Biochemistry (Moscow)
Том: 88
Номер: 9
Год издания: 2023
Издательство: Pleiades Publishing, Ltd
Местоположение издательства: Road Town, United Kingdom
Первая страница: 1378
Последняя страница: 1389
DOI: 10.1134/s0006297923090171
Аннотация: 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.
Добавил в систему: Пометун Евгений Владимирович

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

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