Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure

Boyko, K.M.; Kryukova, M.V.; Petrovskaya, L.E.; Kryukova, E.A.; Nikolaeva, A.Y.; Korzhenevsky, D.A.; Lomakina, G.Y.; Novototskaya-Vlasova, K.A.; Rivkina, E.M.; Dolgikh, D.A.; Kirpichnikov, M.P.; Popov, V.O.

Авторы: Boyko K.M., Kryukova M.V., Petrovskaya L.E., Kryukova E.A., Nikolaeva A.Y., Korzhenevsky D.A., Lomakina G.Yu, Novototskaya-Vlasova K.A., Rivkina E.M., Dolgikh D.A., Kirpichnikov M.P., Popov V.O.
Журнал: Biomolecules
Том: 11
Номер: 1
Год издания: 2021
Издательство: MDPI
Местоположение издательства: Basel, Switzerland
Первая страница: 1
Последняя страница: 18
Номер статьи: 57
DOI: 10.3390/biom11010057
Аннотация: Abstract: The gene coding for a novel cold-active esterase PMGL3 was previously obtained from aSiberian permafrost metagenomic DNA library and expressed in Escherichia coli. We elucidated the3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar toother bacterial HSLs, PMGL3 shares a canonical / hydrolase fold and is presumably a dimer insolution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolongedincubation at 4 C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a uniquearchitecture compared to other known tetramers of the bacterial HSLs. To study the role of thespecific residues comprising the tetramerization interface of PMGL3, several mutant variants wereconstructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutantsdemonstrated that they still contained a portion of tetrameric form after heat treatment, although itsamount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7Nand K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 C in comparison withthe wild type protein. Km values of these mutants were similar to that of the wt PMGL3. However,the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.
Добавил в систему: Ломакина Галина Юрьевна

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Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structureстатья

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