Chimeric versus isolated proteins: Biochemical characterization of the NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 fused with the Baeyer-Villiger monooxygenase from Thermobifida fusca

Lio, E.; Parshin, P.; D'Oronzo, E.; Plebani, S.; Pometun, A.A.; Kleymenov, S.Y.; Tishkov, V.I.; Secundo, F.

Авторы: Lio Elia, Parshin Pavel, D'Oronzo Erica, Plebani Stefano, Pometun Anastasia A., Kleymenov S.Yu, Tishkov Vladimir I., Secundo Francesco
Журнал: International Journal of Biological Macromolecules
Том: 253
Год издания: 2023
Издательство: Elsevier BV
Местоположение издательства: Netherlands
Номер статьи: 126637
DOI: 10.1016/j.ijbiomac.2023.126637
Аннотация: The expression of multifunctional proteins can facilitate the setup of a biotechnology process that requires multiple functions absolved by different proteins. Herein the functional and conformational characterization of a formate dehydrogenase-monooxygenase chimera enzyme is presented. The fused enzyme (FDH-PAMO) wasprepared by linking the C-terminus of the mutant NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 (FDH) to the N-terminus of the NADPH-dependent monooxygenase from Thermobifida fusca (PAMO) through a peptide linker of 9 amino acids (ASGGGGSGT) generating a chimera protein of 107,056 Da. The catalytic properties (e.g., kinetic parameters kcat and Km), stability, fluorescence and circular dichroism spectra showed that the so-obtained chimera enzyme FDH-PAMO retains the same functional and conformational properties of the two parental enzymes. Furthermore, SEC chromatographic analysis indicated that, in solution (pH 7.4), FDH-PAMO assembles to tetramers (up to 4.2 %) due to the propensity of FDH and PAMO to form dimers, up to 96.6 % and 6.2 %, respectively. This study provides valuable insights into the structural stability of a thermostable protein (e.g., PAMO) after increasing its size through fusion with another similarly sized thermostableprotein (e.g., FDH).
Добавил в систему: Тишков Владимир Иванович

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Chimeric versus isolated proteins: Biochemical characterization of the NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 fused with the Baeyer-Villiger monooxygenase from Thermobifida fuscaстатья

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