Publications by authors named "Ksenia A Novototskaya-Vlasova"

4 Publications

  • Page 1 of 1

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

Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine.

PLoS One 2020 28;15(1):e0226838. Epub 2020 Jan 28.

Department of Enzyme Engineering, Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.

Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226838PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986724PMC
April 2020

New member of the hormone-sensitive lipase family from the permafrost microbial community.

Bioengineered 2017 Jul 18;8(4):420-423. Epub 2016 Oct 18.

b Institute of Physicochemical and Biological Problems in Soil Science , Russian Academy of Sciences , Pushchino , Russia.

Siberian permafrost is a unique environment inhabited with diverse groups of microorganisms. Among them, there are numerous producers of biotechnologically relevant enzymes including lipases and esterases. Recently, we have constructed a metagenomic library from a permafrost sample and identified in it several genes coding for potential lipolytic enzymes. In the current work, properties of the recombinant esterases obtained from this library are compared with the previously characterized lipase from Psychrobacter cryohalolentis and other representatives of the hormone-sensitive lipase family.
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http://dx.doi.org/10.1080/21655979.2016.1230571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553336PMC
July 2017

Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

FEMS Microbiol Ecol 2016 May 28;92(5):fiw046. Epub 2016 Feb 28.

Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Institutskaya str., 2, 142290, Pushchino, Moscow Region, Russia.

As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.
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http://dx.doi.org/10.1093/femsec/fiw046DOI Listing
May 2016
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