Publications by authors named "Elena A Kryukova"

3 Publications

  • Page 1 of 1

Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from with Unusual Amino Acid Content.

Biomolecules 2021 08 17;11(8). Epub 2021 Aug 17.

Department of Bioengineering, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium from Siberian permafrost soil was cloned and expressed in . The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops-a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with -nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to "the proline rule". Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min.
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http://dx.doi.org/10.3390/biom11081229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392543PMC
August 2021

Increased Synthesis of a Magnesium Transporter MgtA During Recombinant Autotransporter Expression in Escherichia coli.

Appl Biochem Biotechnol 2021 Nov 5;193(11):3672-3703. Epub 2021 Aug 5.

Shemyakin & Ovchinnikov Institute of Bioorganic , Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya, 16/10, Moscow, 117997, Russia.

Overproduction of the membrane proteins in Escherichia coli cells is a common approach to obtain sufficient material for their functional and structural studies. However, the efficiency of this process can be limited by toxic effects which decrease the viability of the host and lead to low yield of the product. During the expression of the esterase autotransporter AT877 from Psychrobacter cryohalolentis K5, we observed significant growth inhibition of the C41(DE3) cells in comparison with the same cells producing other recombinant proteins. Induction of AT877 synthesis also resulted in the elevated expression of a magnesium transporter MgtA and decreased ATP content of the cells. To characterize the response to overexpression of the autotransporter in bacterial cells, we performed a comparative analysis of their proteomic profile by mass spectrometry. According to the obtained data, E. coli cells which synthesize AT877 experience complex stress condition presumably associated with secretion apparatus overloading and improper localization of the recombinant protein. Several response pathways were shown to be activated by AT877 overproduction including Cpx, PhoP/PhoQ, Psp, and σ The obtained results open new opportunities for optimization of the recombinant membrane protein expression in E. coli for structural studies and biotechnological applications.
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http://dx.doi.org/10.1007/s12010-021-03634-5DOI Listing
November 2021

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

Biomolecules 2021 01 5;11(1). Epub 2021 Jan 5.

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in . 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. 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%.
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http://dx.doi.org/10.3390/biom11010057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824956PMC
January 2021
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