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. 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%.
Highlights
IntroductionLipases and esterases from extremophilic microorganisms (thermophilic, psychrophilic, halophilic, etc.) frequently possess unique structural and functional characteristics which enable them to operate in harsh environmental conditions [3]
Our results demonstrated that mutations of non-conserved D7 and K67 residues mostly affected the tetramerization and the thermal stability of PMGL3
A major difference between GDSAG and GT/XSAG bHSL subfamilies is the mutual orientation of β8 strands within the dimeric interface
Summary
Lipases and esterases from extremophilic microorganisms (thermophilic, psychrophilic, halophilic, etc.) frequently possess unique structural and functional characteristics which enable them to operate in harsh environmental conditions [3]. These properties provide a foundation for the ongoing search for new representatives of the family and numerous studies devoted to their characterization. Many cold-active lipolytic enzymes demonstrate high catalytic efficiency and activity at low temperatures [4,5]. They can catalyze reactions in the presence of high salt concentration and organic solvents. Low thermal stability usually limits their broad industrial application
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
