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

by Konstantin M. Boyko, Mariya V. Kryukova, Lada E. Petrovskaya, Elena A. Kryukova, Alena Y. Nikolaeva, Dmitry A. Korzhenevsky, Galina Yu. Lomakina, Ksenia A. Novototskaya-Vlasova, Elizaveta M. Rivkina, Dmitry A. Dolgikh, Mikhail P. Kirpichnikov and Vladimir O. Popov

Biomolecules 2021, 11(1), 57; https://doi.org/10.3390/biom11010057 - 5 Jan 2021

Cited by 8 | Viewed by 3262

Abstract

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in Escherichia coli. We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/β hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. K_m 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%. Full article

(This article belongs to the Collection Feature Papers in Biochemistry)

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13 pages, 1906 KB

Open AccessArticle

Thermal Inactivation of a Cold-Active Esterase PMGL3 Isolated from the Permafrost Metagenomic Library

by M.V. Kryukova, L.E. Petrovskaya, E.A. Kryukova, G.Yu. Lomakina, S.A. Yakimov, E.G. Maksimov, K.M. Boyko, V.O. Popov, D.A. Dolgikh and M.P. Kirpichnikov

Biomolecules 2019, 9(12), 880; https://doi.org/10.3390/biom9120880 - 16 Dec 2019

Cited by 12 | Viewed by 3392

Abstract

PMGL3 is a cold-adapted esterase which was recently isolated from the permafrost metagenomic library. It exhibits maximum activity at 30 °C and low stability at elevated temperatures (40 °C and higher). Sequence alignment has revealed that PMGL3 is a member of the hormone-sensitive lipase (HSL) family. In this work, we demonstrated that incubation at 40 °C led to the inactivation of the enzyme (t_1/2 = 36 min), which was accompanied by the formation of tetramers and higher molecular weight aggregates. In order to increase the thermal stability of PMGL3, its two cysteines Cys49 and Cys207 were substituted by the hydrophobic residues, which are found at the corresponding positions of thermostable esterases from the HSL family. One of the obtained mutants, C207F, possessed improved stability at 40 °C (t_1/2 = 169 min) and increased surface hydrophobicity, whereas C49V was less stable in comparison with the wild type PMGL3. Both mutants exhibited reduced values of V_max and k_cat, while C207F demonstrated increased affinity to the substrate, and improved catalytic efficiency. Full article

(This article belongs to the Special Issue Protein Folding and Quality Control Mechanisms - In Memory of Prof. Oleg B. Ptitsyn (1929-1999))

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