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

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; M P Kirpichnikov

doi:10.3390/biom9120880

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

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

Authors

M V Kryukova¹, L E Petrovskaya², E A Kryukova², G Yu Lomakina³, S A Yakimov², E G Maksimov⁴, K M Boyko⁵, V O Popov^{1

5}, D A Dolgikh^{2

4}, M P Kirpichnikov^{2

4}

Affiliations

¹ Kurchatov Complex of NBICS-technologies, National Research Centre "Kurchatov Institute", Akad. Kurchatova sqr 1, 123182 Moscow, Russia.
² Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya, 16/10, 117997 Moscow, Russia.
³ Department of Chemistry, Lomonosov Moscow State University, Leninskiye Gory, 1/3, 119991 Moscow, Russia.
⁴ Department of Biology, Lomonosov Moscow State University, Leninskie gory, 1, bld 12, 119234 Moscow, Russia.
⁵ Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2, 119071 Moscow, Russia.

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.

Keywords: HSL family; PMGL3 esterase; cold-active proteins; cysteine mutagenesis; hydrophobicity; stability; thermal inactivation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cold Temperature*
Enzyme Stability
Esterases / antagonists & inhibitors*
Esterases / chemistry
Esterases / isolation & purification*
Esterases / metabolism
Gene Library*
Metagenome / genetics*
Permafrost / microbiology*

Substances

Esterases