Publications by authors named "Konstantin M Boyko"

27 Publications

  • Page 1 of 1

LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift.

Int J Mol Sci 2021 Nov 28;22(23). Epub 2021 Nov 28.

Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 123182 Moscow, Russia.

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs' performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.
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http://dx.doi.org/10.3390/ijms222312887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8657457PMC
November 2021

The Uncommon Active Site of D-Amino Acid Transaminase from : Biochemical and Structural Insights into the New Enzyme.

Molecules 2021 Aug 20;26(16). Epub 2021 Aug 20.

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2, 119071 Moscow, Russia.

Among industrially important pyridoxal-5'-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the relationships in these enzymes.
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http://dx.doi.org/10.3390/molecules26165053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8401098PMC
August 2021

Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.

PLoS One 2021 29;16(7):e0255098. Epub 2021 Jul 29.

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

Creating biocatalysts for (R)-selective amination effectively is highly desirable in organic synthesis. Despite noticeable progress in the engineering of (R)-amine activity in pyridoxal-5'-phosphate-dependent transaminases of fold type IV, the specialization of the activity is still an intuitive task, as there is poor understanding of sequence-structure-function relationships. In this study, we analyzed this relationship in transaminase from Thermobaculum terrenum, distinguished by expanded substrate specificity and activity in reactions with L-amino acids and (R)-(+)-1-phenylethylamine using α-ketoglutarate and pyruvate as amino acceptors. We performed site-directed mutagenesis to create a panel of the enzyme variants, which differ in the active site residues from the parent enzyme to a putative transaminase specific to (R)-primary amines. The variants were examined in the overall transamination reactions and half-reaction with (R)-(+)-1-phenylethylamine. A structural analysis of the most prominent variants revealed a spatial reorganization in the active sites, which caused changes in activity. Although the specialization to (R)-amine transaminase was not implemented, we succeeded in understanding the role of the particular active site residues in expanding substrate specificity of the enzyme. We showed that the specificity for (R)-(+)-1-phenylethylamine in transaminase from T. terrenum arises without sacrificing the specificity for L-amino acids and α-ketoglutarate and in consensus with it.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0255098PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8320979PMC
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

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity.

Int J Mol Sci 2020 Apr 24;21(8). Epub 2020 Apr 24.

Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 123182 Moscow, Russia.

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi and , which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.
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http://dx.doi.org/10.3390/ijms21083012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215472PMC
April 2020

Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein.

Int J Mol Sci 2020 Feb 28;21(5). Epub 2020 Feb 28.

National Research Center "Kurchatov Institute", Moscow 123182, Russia.

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (K of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively.
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http://dx.doi.org/10.3390/ijms21051644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084697PMC
February 2020

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

Structural insight into the substrate specificity of PLP fold type IV transaminases.

Appl Microbiol Biotechnol 2020 Mar 27;104(6):2343-2357. Epub 2020 Jan 27.

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2, Moscow, Russian Federation, 119071.

Pyridoxal-5'-phosphate-dependent transaminases of fold type IV (class IV) are promising enzymes for (R)-selective amination of organic compounds. Transaminases of fold type IV exhibit either strict (R)-selectivity or (S)-selectivity that is implemented within geometrically similar active sites of different amino acid compositions. Based on substrate specificity, class IV comprises three large families of transaminases: (S)-selective branched-chain L-amino acid aminotransferases and (R)-selective D-amino acid aminotransferases and (R)-amine:pyruvate transaminases. In this review, we aim to analyze the substrate profiles and correlations between the substrate specificity and organization of the active site in transaminases from these structurally related families. New transaminases with an expanded substrate specificity are also discussed. An analysis of the structural features of substrate binding and comparisons of structural determinants of chiral discrimination between members of the class IV transaminases could be helpful in identifying new biocatalytically relevant enzymes as well as rational protein engineering.
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http://dx.doi.org/10.1007/s00253-020-10369-6DOI Listing
March 2020

Identification, functional and structural characterization of novel aminoglycoside phosphotransferase APH(3″)-Id from Streptomyces rimosus subsp. rimosus ATCC 10970.

Arch Biochem Biophys 2019 08 26;671:111-122. Epub 2019 Jun 26.

Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991, Russia.

In this study, we identified a new gene (aph(3″)-Id) coding for a streptomycin phosphotransferase by using phylogenetic comparative analysis of the genome of the oxytetracycline-producing strain Streptomyces rimosus ATCC 10970. Cloning the aph(3″)-Id gene in E.coli and inducing its expression led to an increase in the minimum inhibitory concentration of the recombinant E.coli strain to streptomycin reaching 350 μg/ml. To evaluate the phosphotransferase activity of the recombinant protein APH(3″)-Id we carried out thin-layer chromatography of the putative P-labeled streptomycin phosphate. We also performed a spectrophotometric analysis to determine the production of ADP coupled to NADH oxidation. Here are the kinetic parameters of the streptomycin phosphotransferase APH(3″)-Id: K 80.4 μM, V 6.45 μmol/min/mg and k 1.73 s. We demonstrated for the first time the ability of the aminoglycoside phototransferase (APH(3″)-Id) to undergo autophosphorylation in vitro. The 3D structures of APH(3″)-Id in its unliganded state and in ternary complex with streptomycin and ADP were obtained. The structure of the ternary complex is the first example of this class of enzymes with bound streptomycin. Comparison of the obtained structures with those of other aminoglycoside phosphotransferases revealed peculiar structure of the substrate-binding pocket reflecting its specificity to a particular antibiotic.
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http://dx.doi.org/10.1016/j.abb.2019.06.008DOI Listing
August 2019

Functional characterization of PLP fold type IV transaminase with a mixed type of activity from Haliangium ochraceum.

Biochim Biophys Acta Proteins Proteom 2019 06 19;1867(6):575-585. Epub 2019 Mar 19.

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2, 119071 Moscow, Russian Federation.

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are industrially important enzymes catalyzing the stereoselective amination of ketones and keto acids. Transaminases of PLP fold type IV are characterized by (R)- or (S)-stereoselective transfer of amino groups, depending on the substrate profile of the enzyme. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases and (R)-amine:pyruvate transaminases. Recently, transaminases with a mixed type of activity were identified and characterized. Here, we report biochemical and structural characterization of a transaminase from myxobacterium Haliangium ochraceum (Hoch3033), which is active towards keto analogs of branched-chain amino acids (specific substrates for BCATs) and (R)-(+)-α-methylbenzylamine (specific substrate for (R)-amine:pyruvate transaminases). The enzyme is characterized by an alkaline pH optimum (pH 10.0-10.5) and a tolerance to high salt concentrations (up to 2 M NaCl). The structure of Hoch3033 was determined at 2.35 Å resolution. The overall fold of the enzyme was similar to those of known enzymes of PLP fold type IV. The mixed type of activity of Hoch3033 was implemented within the BCAT-like active site. However, in the active site of Hoch3033, we observed substitutions of specificity-determining residues that are important for substrate binding in canonical BCATs. We suggest that these changes result in the loss of activity towards α-ketoglutarate and increase the affinity towards (R)-(+)-α-methylbenzylamine. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further research to understand the structural basis of substrate specificity in these enzymes.
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http://dx.doi.org/10.1016/j.bbapap.2019.03.005DOI Listing
June 2019

Thermostable Branched-Chain Amino Acid Transaminases From the Archaea and : Biochemical and Structural Characterization.

Front Bioeng Biotechnol 2019 24;7. Epub 2019 Jan 24.

Henry Wellcome Building for Biocatalysis, Biosciences, University of Exeter, Exeter, United Kingdom.

Two new thermophilic branched chain amino acid transaminases have been identified within the genomes of different hyper-thermophilic archaea, , and . These enzymes belong to the class IV of transaminases as defined by their structural fold. The enzymes have been cloned and over-expressed in and the recombinant enzymes have been characterized both biochemically and structurally. Both enzymes showed high thermostability with optimal temperature for activity at 80 and 85°C, respectively. They retain good activity after exposure to 50% of the organic solvents, ethanol, methanol, DMSO and acetonitrile. The enzymes show a low activity to ()-methylbenzylamine but no activity to ()-methylbenzylamine. Both enzymes have been crystallized and their structures solved in the internal aldimine form, to 1.9 Å resolution for the enzyme and 2.0 Å for the enzyme. Also the enzyme structure has been determined in complex with the amino acceptor α-ketoglutarate and the enzyme in complex with the inhibitor gabaculine. These two complexes have helped to determine the conformation of the enzymes during enzymatic turnover and have increased understanding of their substrate specificity. A comparison has been made with another () selective class IV transaminase from the fungus which was previously studied in complex with gabaculine. The subtle structural differences between these enzymes has provided insight regarding their different substrate specificities.
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http://dx.doi.org/10.3389/fbioe.2019.00007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353796PMC
January 2019

Biochemical and structural insights into PLP fold type IV transaminase from Thermobaculum terrenum.

Biochimie 2019 Mar 29;158:130-138. Epub 2018 Dec 29.

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, Bld. 2, 119071, Moscow, Russian Federation; Kurchatov Complex of NBICS-technologies, National Research Centre "Kurchatov Institute", Akad. Kurchatova Sqr 1, 123182, Moscow, Russian Federation.

The high catalytic efficiency of enzymes under reaction conditions is one of the main goals in biocatalysis. Despite the dramatic progress in the development of more efficient biocatalysts by protein design, the search for natural enzymes with useful properties remains a promising strategy. The pyridoxal 5'-phosphate (PLP)-dependent transaminases represent a group of industrially important enzymes due to their ability to stereoselectively transfer amino groups between diverse substrates; however, the complex mechanism of substrate recognition and conversion makes the design of transaminases a challenging task. Here we report a detailed structural and kinetic study of thermostable transaminase from the bacterium Thermobaculum terrenum (TaTT) using the methods of enzyme kinetics, X-ray crystallography and molecular modeling. TaTT can convert L-branched-chain and L-aromatic amino acids as well as (R)-(+)-1-phenylethylamine at a high rate and with high enantioselectivity. The structures of TaTT in complex with the cofactor pyridoxal 5'-phosphate covalently bound to enzyme and in complex with its reduced form, pyridoxamine 5'-phosphate, were determined at resolutions of 2.19 Å and 1.5 Å, and deposited in the Protein Data Bank as entries 6GKR and 6Q8E, respectively. TaTT is a fold type IV PLP-dependent enzyme. In terms of structural similarity, the enzyme is close to known branched-chain amino acid aminotransferases, but differences in characteristic sequence motifs in the active site were observed in TaTT compared to canonical branched-chain amino acid aminotransferases, which can explain the improved binding of aromatic amino acids and (R)-(+)-1-phenylethylamine. This study has shown for the first time that high substrate specificity towards both various l-amino acids and (R)-primary amines can be implemented within one pyridoxal 5'-phosphate-dependent active site of fold type IV. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further biochemical and bioinformatic studies to understand the sequence-structure-function relationship in these enzymes.
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http://dx.doi.org/10.1016/j.biochi.2018.12.017DOI Listing
March 2019

3D structure of the natural tetrameric form of human butyrylcholinesterase as revealed by cryoEM, SAXS and MD.

Biochimie 2019 Jan 29;156:196-205. Epub 2018 Oct 29.

National Research Center, Kurchatov Institute, Akademika Kurchatova pl. 1, Moscow, 123182, Russia; Shubnikov Institute of Crystallography of Federal Scientific Research Centre "Crystallography and Photonics" Russian Academy of Sciences, Leninsky pr. 59, Moscow, 119333, Russia.

Human plasma butyrylcholinesterase (BChE) is an endogenous bioscavenger that hydrolyzes numerous medicamentous and poisonous esters and scavenges potent organophosphorus nerve agents. BChE is thus a marker for the diagnosis of OP poisoning. It is also considered a therapeutic target against Alzheimer's disease. Although the X-ray structure of a partially deglycosylated monomer of human BChE was solved 15 years ago, all attempts to determine the 3D structure of the natural full-length glycosylated tetrameric human BChE have been unsuccessful so far. Here, a combination of three complementary structural methods-single-particle cryo-electron microscopy, molecular dynamics and small-angle X-ray scattering-were implemented to elucidate the overall structural and spatial organization of the natural tetrameric human plasma BChE. A 7.6 Å cryoEM map clearly shows the major features of the enzyme: a dimer of dimers with a nonplanar monomer arrangement, in which the interconnecting super helix complex PRAD-(WAT)-peptide C-terminal tail is located in the center of the tetramer, nearly perpendicular to its plane, and is plunged deep between the four subunits. Molecular dynamics simulations allowed optimization of the geometry of the molecule and reconstruction of the structural features invisible in the cryoEM density, i.e., glycan chains and glycan interdimer contact areas, as well as intermonomer disulfide bridges at the C-terminal tail. Finally, SAXS data were used to confirm the consistency of the obtained model with the experimental data. The tetramer organization of BChE is unique in that the four subunits are joined at their C-termini through noncovalent contacts with a short polyproline-rich peptide. This tetramer structure could serve as a model for the design of highly stable glycosylated tetramers.
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http://dx.doi.org/10.1016/j.biochi.2018.10.017DOI Listing
January 2019

Diaminopelargonic acid transaminase from Psychrobacter cryohalolentis is active towards (S)-(-)-1-phenylethylamine, aldehydes and α-diketones.

Appl Microbiol Biotechnol 2018 Nov 3;102(22):9621-9633. Epub 2018 Sep 3.

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2, Moscow, Russian Federation, 119071.

Substrate and reaction promiscuity is a remarkable property of some enzymes and facilitates the adaptation to new metabolic demands in the evolutionary process. Substrate promiscuity is also a basis for protein engineering for biocatalysis. However, molecular principles of enzyme promiscuity are not well understood. Even for the widely studied PLP-dependent transaminases of class III, the reliable prediction of the biocatalytically important amine transaminase activity is still difficult if the desired activity is unrelated to the natural activity. Here, we show that 7,8-diaminopelargonic acid transaminase (synthase), previously considered to be highly specific, is able to convert (S)-(-)-1-phenylethylamine and a number of aldehydes and diketones. We were able to characterize the (S)-amine transaminase activity of 7,8-diaminopelargonic acid transaminase from Psychrobacter cryohalolentis (Pcryo361) and analyzed the three-dimensional structure of the enzyme. New substrate specificity for α-diketones was observed, though only a weak activity towards pyruvate was found. We examined the organization of the active site and binding modes of S-adenosyl-L-methionine and (S)-(-)-1-phenylethylamine using X-ray analysis and molecular docking. We suggest that the Pcryo361 affinity towards (S)-(-)-1-phenylethylamine arises from the recognition of the hydrophobic parts of the specific substrates, S-adenosyl-L-methionine and 7-keto-8-aminopelargonic acid, and from the flexibility of the active site. Our results support the observation that the conversion of amines is a promiscuous activity of many transaminases of class III and is independent from their natural function. The analysis of amine transaminase activity from among various transaminases will help to make the sequence-function prediction for biocatalysis more reliable.
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http://dx.doi.org/10.1007/s00253-018-9310-0DOI Listing
November 2018

Structural characterization of geranylgeranyl pyrophosphate synthase GACE1337 from the hyperthermophilic archaeon Geoglobus acetivorans.

Extremophiles 2018 Nov 30;22(6):877-888. Epub 2018 Jul 30.

Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, Bld. 2, Moscow, 119071, Russian Federation.

A novel type 1 geranylgeranyl pyrophosphate synthase GACE1337 has been identified within the genome of a newly identified hyperthermophilic archaeon Geoglobus acetivorans. The enzyme has been cloned and over-expressed in Escherichia coli. The recombinant enzyme has been biochemically and structurally characterized. It is able to catalyze the synthesis of geranylgeranyl pyrophosphate as a major product and of farnesyl pyrophosphate in smaller amounts, as measured by gas chromatography-mass spectrometry at an elevated temperature of 60 °C. Its ability to produce two products is consistent with the fact that GACE1337 is the only short-chain isoprenyl diphosphate synthase in G. acetivorans. Attempts to crystallize the enzyme were successful only at 37 °C. The three-dimensional structure of GACE1337 was determined by X-ray diffraction to 2.5 Å resolution. A comparison of its structure with those of related enzymes revealed that the Geoglobus enzyme has the features of both type I and type III geranylgeranyl pyrophosphate synthases, which allow it to regulate the product length. The active enzyme is a dimer and has three aromatic amino acids, two Phe, and a Tyr, located in the hydrophobic cleft between the two subunits. It is proposed that these bulky residues play a major role in the synthetic reaction by controlling the product elongation.
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http://dx.doi.org/10.1007/s00792-018-1044-5DOI Listing
November 2018

Structure of an Broad-Range Prophage Endolysin Reveals a C-Terminal α-Helix with the Proposed Role in Activity against Live Bacterial Cells.

Viruses 2018 06 6;10(6). Epub 2018 Jun 6.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.

Proteins that include enzymatic domain degrading the bacterial cell wall and a domain providing transport through the bacterial outer membrane are considered as prospective compounds to combat pathogenic Gram-negative bacteria. This paper presents an isolation and study of an enzyme of this class naturally encoded in the prophage region of AB 5075 genome. Recombinant protein expressed in exhibits an antimicrobial activity with respect to live cultures of Gram-negative bacteria reducing the population of viable bacteria by 1.5⁻2 log colony forming units (CFU)/mL. However the protein becomes rapidly inactivated and enables the bacteria to restore the population. AcLys structure determined by X-ray crystallography reveals a predominantly α—helical fold similar to bacteriophage P22 lysozyme. The С-terminal part of AcLys polypeptide chains forms an α—helix enriched by Lys and Arg residues exposed outside of the protein globule. Presumably this type of structure of the C-terminal α—helix has evolved evolutionally enabling the endolysin to pass the inner membrane during the host lysis or, potentially, to penetrate the outer membrane of the Gram-negative bacteria.
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http://dx.doi.org/10.3390/v10060309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6024848PMC
June 2018

Comparison of histone-like HU protein DNA-binding properties and HU/IHF protein sequence alignment.

PLoS One 2017 13;12(11):e0188037. Epub 2017 Nov 13.

Kurchatov Complex of NBICS-Technologies, National Research Center «Kurchatov Institute», Moscow, Russian Federation.

Background: The structure and function of bacterial nucleoid are controlled by histone-like proteins of HU/IHF family, omnipresent in bacteria and also founding archaea and some eukaryotes.HU protein binds dsDNA without sequence specificity and avidly binds DNA structures with propensity to be inclined such as forks, three/four-way junctions, nicks, overhangs and DNA bulges. Sequence comparison of thousands of known histone-like proteins from diverse bacteria phyla reveals relation between HU/IHF sequence, DNA-binding properties and other protein features.

Methodology And Principal Findings: Performed alignment and clusterization of the protein sequences show that HU/IHF family proteins can be unambiguously divided into three groups, HU proteins, IHF_A and IHF_B proteins. HU proteins, IHF_A and IHF_B proteins are further partitioned into several clades for IHF and HU; such a subdivision is in good agreement with bacterial taxonomy. We also analyzed a hundred of 3D fold comparative models built for HU sequences from all revealed HU clades. It appears that HU fold remains similar in spite of the HU sequence variations. We studied DNA-binding properties of HU from N. gonorrhoeae, which sequence is similar to one of E.coli HU, and HU from M. gallisepticum and S. melliferum which sequences are distant from E.coli protein. We found that in respect to dsDNA binding, only S. melliferum HU essentially differs from E.coli HU. In respect to binding of distorted DNA structures, S. melliferum HU and E.coli HU have similar properties but essentially different from M. gallisepticum HU and N. gonorrhea HU. We found that in respect to dsDNA binding, only S. melliferum HU binds DNA in non-cooperative manner and both mycoplasma HU bend dsDNA stronger than E.coli and N. gonorrhoeae. In respect to binding to distorted DNA structures, each HU protein has its individual profile of affinities to various DNA-structures with the increased specificity to DNA junction.

Conclusions And Significance: HU/IHF family proteins sequence alignment and classification are updated. Comparative modeling demonstrates that HU protein 3D folding's even more conservative than HU sequence. For the first time, DNA binding characteristics of HU from N. gonorrhoeae, M. gallisepticum and S. melliferum are studied. Here we provide detailed analysis of the similarity and variability of DNA-recognizing and bending of four HU proteins from closely and distantly related HU clades.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188037PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5683647PMC
December 2017

NADP-Dependent Aldehyde Dehydrogenase from Archaeon : Structural and Functional Features.

Archaea 2016 10;2016:9127857. Epub 2016 Nov 10.

A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, Bld. 2, Moscow 119071, Russia; NBICS Center, National Research Centre "Kurchatov Institute", Akad. Kurchatova Sqr. 1, Moscow 123182, Russia.

We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. , AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60-85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution), three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å), and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å). The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues) and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.
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http://dx.doi.org/10.1155/2016/9127857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5121451PMC
July 2017

Structural basis of the high thermal stability of the histone-like HU protein from the mollicute Spiroplasma melliferum KC3.

Sci Rep 2016 11 3;6:36366. Epub 2016 Nov 3.

Kurchatov Complex of NBICS-Technologies, National Research Center "Kurchatov Institute", Akad. Kurchatova pl., 1, Moscow, 123182, Russian Federation.

The three-dimensional structure of the histone-like HU protein from the mycoplasma Spiroplasma melliferum KC3 (HUSpm) was determined at 1.4 Å resolution, and the thermal stability of the protein was evaluated by differential scanning calorimetry. A detailed analysis revealed that the three-dimensional structure of the HUSpm dimer is similar to that of its bacterial homologues but is characterized by stronger hydrophobic interactions at the dimer interface. This HUSpm dimer interface lacks salt bridges but is stabilized by a larger number of hydrogen bonds. According to the DSC data, HUSpm has a high denaturation temperature, comparable to that of HU proteins from thermophilic bacteria. To elucidate the structural basis of HUSpm thermal stability, we identified amino acid residues potentially responsible for this property and modified them by site-directed mutagenesis. A comparative analysis of the melting curves of mutant and wild-type HUSpm revealed the motifs that play a key role in protein thermal stability: non-conserved phenylalanine residues in the hydrophobic core, an additional hydrophobic loop at the N-terminal region of the protein, the absence of the internal cavity present at the dimer interface of some HU proteins, and the presence of additional hydrogen bonds between the monomers that are missing in homologous proteins.
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http://dx.doi.org/10.1038/srep36366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5093408PMC
November 2016

Structural characterization of the novel aminoglycoside phosphotransferase AphVIII from Streptomyces rimosus with enzymatic activity modulated by phosphorylation.

Biochem Biophys Res Commun 2016 09 20;477(4):595-601. Epub 2016 Jun 20.

Bach Institute of Biochemistry, Federal Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky Prospekt. 33, Bld. 2, 119071, Moscow, Russian Federation; National Research Center "Kurchatov Institute", Kurchatov Complex of NBICS-technologies, Akad. Kurchatova sqr., 1, Moscow, 123182, Russian Federation.

Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3'-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 Å. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop with unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding.
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http://dx.doi.org/10.1016/j.bbrc.2016.06.097DOI Listing
September 2016

First structure of archaeal branched-chain amino acid aminotransferase from Thermoproteus uzoniensis specific for L-amino acids and R-amines.

Extremophiles 2016 Mar 12;20(2):215-25. Epub 2016 Feb 12.

A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2, Moscow, 119071, Russian Federation.

The gene TUZN1299 from the genome of the hyperthermophilic archaeon Thermoproteus uzoniensis encoding a new 32.8 kDa branched-chain amino acid aminotransferase (BCAT) was expressed in Escherichia coli. The recombinant protein TUZN1299 was purified to homogeneity in the PLP-bound form. TUZN1299 was active towards branched-chain amino acids (L-Val, L-Leu, L-Ile) and showed low but detectable activity toward (R)-alpha-methylbenzylamine. The enzyme exhibits high-temperature optimum, thermal stability, and tolerance to organic solvents. The structure of an archaeal BCAT called TUZN1299 was solved for the first time (at 2.0 Å resolution). TUZN1299 has a typical BCAT type IV fold, and the organization of its active site is similar to that of bacterial BCATs. However, there are some differences in the amino acid composition of the active site.
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http://dx.doi.org/10.1007/s00792-016-0816-zDOI Listing
March 2016

Structural insight into the molecular basis of polyextremophilicity of short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus.

Biochimie 2012 Dec 5;94(12):2628-38. Epub 2012 Aug 5.

A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Pr. 33, Moscow 119071, Russia.

Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme - half-life of 1 h at 100 °C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 Å resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate + glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous "resilience" of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.
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http://dx.doi.org/10.1016/j.biochi.2012.07.024DOI Listing
December 2012

Covalent modifications of the catalytic tyrosine in octahaem cytochrome c nitrite reductase and their effect on the enzyme activity.

Acta Crystallogr D Biol Crystallogr 2012 Feb 13;68(Pt 2):144-53. Epub 2012 Jan 13.

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russian Federation.

Octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR), like the previously characterized pentahaem nitrite reductases (NrfAs), catalyzes the six-electron reductions of nitrite to ammonia and of sulfite to sulfide. The active site of both TvNiR and NrfAs is formed by the lysine-coordinated haem and His, Tyr and Arg residues. The distinguishing structural feature of TvNiR is the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity of TvNiR compared with NrfAs. In the present study, a new modified form of the enzyme (TvNiRb) that contains an additional covalent bond between Tyr303 CE1 and Gln360 CG is reported. Structures of TvNiRb in complexes with phosphate (1.45 Å resolution) and sulfite (1.8 Å resolution), the structure of TvNiR in a complex with nitrite (1.83 Å resolution) and several additional structures were determined. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site, which is absent in NrfAs. This is an additional argument in favour of the involvement of Tyr303 as a proton donor in catalysis. The changes in the activity of cytochrome c nitrite reductases owing to the formation of Tyr-Cys and Tyr-Gln bonds may be associated with changes in the pK(a) value of the catalytic tyrosine.
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http://dx.doi.org/10.1107/S0907444911052632DOI Listing
February 2012

Structures of complexes of octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens with sulfite and cyanide.

Acta Crystallogr D Biol Crystallogr 2010 Oct 18;66(Pt 10):1043-7. Epub 2010 Sep 18.

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russia.

The structures of complexes of octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens (TvNiR) with the substrate sulfite (1.4 Å resolution; R(cryst) = 0.126) and the inhibitor cyanide (1.55 Å resolution; R(cryst) = 0.148) have been established. The complex with sulfite was prepared by the reduction of the protein crystal with sodium dithionite. The sulfite ion is bound to the iron ion of the catalytic haem through the S atom. The Fe-S distance is 2.24 Å. The structure of the cyanide complex with full occupancy of the ligand site was established for the first time for cytochrome c nitrite reductases. The cyanide ion is bound to the catalytic haem iron through the C atom. The Fe-C distance is 1.91 Å and the Fe-C-N angle is 171°. The sulfite reductase activity of TvNiR was measured at different pH values. The activity is 0.02 µmol of HS(-) per minute per milligram at pH 7.0; it decreases with increasing pH and is absent at pH 9.0.
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http://dx.doi.org/10.1107/S0907444910031665DOI Listing
October 2010

High-resolution structural analysis of a novel octaheme cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens.

J Mol Biol 2009 Jun 23;389(5):846-62. Epub 2009 Apr 23.

A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia.

Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes-the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca(2+)-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.
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http://dx.doi.org/10.1016/j.jmb.2009.04.037DOI Listing
June 2009

Molecular and catalytic properties of a novel cytochrome c nitrite reductase from nitrate-reducing haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens.

Biochim Biophys Acta 2006 Apr 9;1764(4):715-23. Epub 2006 Feb 9.

Bach Institute of Biochemistry, Leninsky pr. 33, 119071 Moscow, Russia.

A highly active cytochrome c nitrite reductase from the haloalkaliphilic sulfur-oxidizing non-ammonifying bacterium Tv. nitratireducens strain ALEN 2 (TvNiR) was isolated and purified to apparent electrophoretic homogeneity. The enzyme catalyzes reductive conversion of nitrite and hydroxylamine to ammonia without release of any intermediates, as well as reduction of sulfite to sulfide. TvNiR also possesses peroxidase activity. In solution TvNiR exists as a stable hexamer with molecular mass of about 360kDa. Each TvNiR subunit with molecular mass of 64kDa contains, as defined from spectral properties and sequence analysis, eight c-type haems. Seven of them are coordinated by the characteristic CXXCH motifs for haem c binding, while one is bonded by the unique CXXCK motif. So far, this motif coordinating the catalytic haem was found only in bacterial cytochrome c nitrite reductases (ccNiRs). All the residues essential for catalysis in the known ccNiRs were also identified in TvNiR. However, TvNiR is only distantly related to known bacterial ammonifying dissimilatory ccNiRs, sharing no more than 20% homology.
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http://dx.doi.org/10.1016/j.bbapap.2005.12.021DOI Listing
April 2006
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