Publications by authors named "Vladimir O Popov"

59 Publications

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

Catalytic Properties of Flavocytochrome c Sulfide Dehydrogenase from Haloalkaliphilic Bacterium Thioalkalivibrio paradoxus.

Biochemistry (Mosc) 2021 Mar;86(3):361-369

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

Flavocytochrome c sulfide dehydrogenase (FCC) is one of the central enzymes of the respiratory chain in sulfur-oxidizing bacteria. FCC catalyzes oxidation of sulfide and polysulfide ions to elemental sulfur accompanied by electron transfer to cytochrome c. The catalytically active form of the enzyme is a non-covalently linked heterodimer composed of flavin- and heme-binding subunits. The Thioalkalivibrio paradoxus ARh1 genome contains five copies of genes encoding homologous FCCs with an amino acid sequence identity from 36 to 54%. When growing on thiocyanate or thiosulfate as the main energy source, the bacterium synthesizes products of different copies of FCC genes. In this work, we isolated and characterized FCC synthesized during the growth of Tv. paradoxus on thiocyanate. FCC was shown to oxidize exclusively sulfide but not other reduced sulfur compounds, such as thiosulfate, sulfite, tetrathionate, and sulfur, and it also does not catalyze the reverse reaction of sulfur reduction to sulfide. Kinetic parameters of the sulfide oxidation reaction are characterized.
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http://dx.doi.org/10.1134/S0006297921030111DOI Listing
March 2021

Novel Extracellular Electron Transfer Channels in a Gram-Positive Thermophilic Bacterium.

Front Microbiol 2020 11;11:597818. Epub 2021 Jan 11.

Winogradsky Institute of Microbiology, FRC Biotechnology Russian Academy of Sciences, Moscow, Russia.

Biogenic transformation of Fe minerals, associated with extracellular electron transfer (EET), allows microorganisms to exploit high-potential refractory electron acceptors for energy generation. EET-capable thermophiles are dominated by hyperthermophilic archaea and Gram-positive bacteria. Information on their EET pathways is sparse. Here, we describe EET channels in the thermophilic Gram-positive bacterium that drive exoelectrogenesis and rapid conversion of amorphous mineral ferrihydrite to large magnetite crystals. Microscopic studies indicated biocontrolled formation of unusual formicary-like ultrastructure of the magnetite crystals and revealed active colonization of anodes in bioelectrochemical systems (BESs) by . The internal structure of micron-scale biogenic magnetite crystals is reported for the first time. Genome analysis and expression profiling revealed three constitutive -type multiheme cytochromes involved in electron exchange with ferrihydrite or an anode, sharing insignificant homology with previously described EET-related cytochromes thus representing novel determinants of EET. Our studies identify these cytochromes as extracellular and reveal potentially novel mechanisms of cell-to-mineral interactions in thermal environments.
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http://dx.doi.org/10.3389/fmicb.2020.597818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829351PMC
January 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

Effects of pH and temperature on (S)-amine activity of transaminase from the cold-adapted bacterium Psychrobacter cryohalolentis.

Extremophiles 2020 Jul 16;24(4):537-549. Epub 2020 May 16.

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

(7R,8S)-diaminopelargonic acid transaminase from the cold-adapted Gram-negative bacterium Psychrobacter cryohalolentis (Pcryo361) is able to react with unnatural substrates including (S)-( +)-1-phenylethylamine, aldehydes and α-diketones. Additionally, Pcryo361 is active at 0-50 °C and retains up to 10% of the maximum activity at 0 °C. Here, we report a detailed study on the stability and low temperature activity of Pcryo361. At the optimal pH for (S)-amine activity (pH 10.0), the enzyme was stable at 0-10 °C and no decrease in the enzyme activity was observed within 24 h in a slightly alkaline medium, pH 8.0, at 35 °C. Pcryo361 was solvent stable and was activated in 10% DMSO and DMFA at 35 °C. An analysis of the efficiency of catalysis of Pcryo361 at 35 °C and 10 °C showed that the specificity towards (S)-( +)-1-phenylethylamine dropped at 10 °C; however, the specificity towards 2,3-butanedione remained unchanged. Inhibition analysis showed that Pcryo361 activity was not inhibited by acetophenone but inhibited by amines (products of aldehyde amination). The observed pH stability and low temperature activity of Pcryo361 with activated keto substrates are attractive features in the field of development of stereoselective amination at low temperatures.
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http://dx.doi.org/10.1007/s00792-020-01174-0DOI Listing
July 2020

Trinuclear copper biocatalytic center forms an active site of thiocyanate dehydrogenase.

Proc Natl Acad Sci U S A 2020 03 24;117(10):5280-5290. Epub 2020 Feb 24.

Research Centre of Biotechnology, Russian Academy of Sciences, 119071 Moscow, Russia;

Biocatalytic copper centers are generally involved in the activation and reduction of dioxygen, with only few exceptions known. Here we report the discovery and characterization of a previously undescribed copper center that forms the active site of a copper-containing enzyme thiocyanate dehydrogenase (suggested EC 1.8.2.7) that was purified from the haloalkaliphilic sulfur-oxidizing bacterium of the genus ubiquitous in saline alkaline soda lakes. The copper cluster is formed by three copper ions located at the corners of a near-isosceles triangle and facilitates a direct thiocyanate conversion into cyanate, elemental sulfur, and two reducing equivalents without involvement of molecular oxygen. A molecular mechanism of catalysis is suggested based on high-resolution three-dimensional structures, electron paramagnetic resonance (EPR) spectroscopy, quantum mechanics/molecular mechanics (QM/MM) simulations, kinetic studies, and the results of site-directed mutagenesis.
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http://dx.doi.org/10.1073/pnas.1922133117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071890PMC
March 2020

Glycated albumin stimulates expression of inflammatory cytokines in muscle cells.

Cytokine 2020 04 27;128:154991. Epub 2020 Jan 27.

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

The effects of glycated albumin on the expression of inflammatory cytokines in differentiated myotubes were investigated. Glycated albumin stimulates the expression of TNF α, IL-1β, IL-6 and CCL-2 both at the mRNA and protein levels via the receptor of AGEs. Various cytokines demonstrated different kinetics of stimulation by glycated albumin. At a high glucose concentration, the stimulation effect was more pronounced than at a low one. At physiological concentrations of albumin and fructosamine, the stimulation effect of glycated albumin on inflammatory cytokine expression in myotubes was also observed. The induction of expression of all studied cytokines was sensitive to the inhibitors of JNK, p38 MAPK, MEK1/2, Src family protein kinases and NF-κB. At the same time, the induction of TNFα and IL-1β was diminished by the Ca/calmodulin-dependent protein kinase inhibitor, whereas the induction of IL-6 and CCL-2 was reduced by the inhibitor of phosphoinositide 3-kinase. Possible implications of observed stimulation of cytokine expression by glycated albumin in the development of diabetes mellitus symptoms are discussed.
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http://dx.doi.org/10.1016/j.cyto.2020.154991DOI Listing
April 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

Comparative Genomics of HRh1 and sp. SCN-R1, Halophilic Chemolithoautotrophic Sulfur-Oxidizing Capable of Using Thiocyanate as Energy Source.

Front Microbiol 2019 1;10:898. Epub 2019 May 1.

Microbial Systems Ecology, Department of Freshwater and Marine Ecology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, Netherlands.

The genomes of and (formerly known as ) sp. SCN-R1, two gammaproteobacterial halophilic sulfur-oxidizing bacteria (SOB) capable of thiocyanate oxidation via the "cyanate pathway", have been analyzed with a particular focus on their thiocyanate-oxidizing potential and sulfur oxidation pathways. Both genomes encode homologs of the enzyme thiocyanate dehydrogenase (TcDH) that oxidizes thiocyanate via the "cyanate pathway" in members of the haloalkaliphilic SOB of the genus However, despite the presence of conservative motives indicative of TcDH, the putative TcDH of the halophilic SOB have a low overall amino acid similarity to the enzyme, and also the surrounding genes in the TcDH locus were different. In particular, an alternative copper transport system is present instead of and a putative zero-valent sulfur acceptor protein gene appears just before TcDH. Moreover, in contrast to the thiocyanate-oxidizing species, both genomes of the halophilic SOB contained a gene encoding the enzyme cyanate hydratase. The sulfur-oxidizing pathway in the genome of includes a Fcc type of sulfide dehydrogenase, a rDsr complex/AprAB/Sat for oxidation of zero-valent sulfur to sulfate, and an incomplete Sox pathway, lacking SoxCD. The sulfur oxidation pathway reconstructed from the genome of sp. SCN-R1 was more similar to that of members of the group, including a Fcc type of sulfide dehydrogenase and a complete Sox complex. One of the outstanding properties of is the presence of a Na-dependent ATP synthase, which is rarely found in aerobic Prokaryotes.Overall, the results showed that, despite an obvious difference in the general sulfur-oxidation pathways, halophilic and haloalkaliphilic SOB belonging to different genera within the developed a similar unique thiocyanate-degrading mechanism based on the direct oxidative attack on the sulfane atom of thiocyanate.
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http://dx.doi.org/10.3389/fmicb.2019.00898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504805PMC
May 2019

Potassium chloride released from contracting skeletal muscle may stimulate development of its hypertrophy.

Biochem Biophys Rep 2019 Jul 21;18:100627. Epub 2019 Mar 21.

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

The effects of potassium chloride on the expression of IGF-1 splice forms and myoblast proliferation were investigated. KCl at the concentrations of 7-12 mM stimulated the synthesis of IGF-1 and mechano growth factor (MGF) in murine myoblasts as well as in myotubes both at the mRNA and protein levels. Pan-calcium channel blocker CdCl completely abolished stimulation of growth factor expression, whereas blocker of HCN and Na1.4 channels ZD7288 drastically reduced it. In addition, potassium chloride stimulated myoblast proliferation, while IGF-1 autocrine signaling inhibition partially suppressed these mitogenic effects.
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http://dx.doi.org/10.1016/j.bbrep.2019.100627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433999PMC
July 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 the flavocytochrome c sulfide dehydrogenase associated with the copper-binding protein CopC from the haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio paradoxusARh 1.

Acta Crystallogr D Struct Biol 2018 07 8;74(Pt 7):632-642. Epub 2018 Jun 8.

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

Flavocytochrome c sulfide dehydrogenase from Thioalkalivibrio paradoxus (TpFCC) is a heterodimeric protein consisting of flavin- and monohaem c-binding subunits. TpFCC was co-purified and co-crystallized with the dimeric copper-binding protein TpCopC. The structure of the TpFCC-(TpCopC) complex was determined by X-ray diffraction at 2.6 Å resolution. The flavin-binding subunit of TpFCC is structurally similar to those determined previously, and the structure of the haem-binding subunit is similar to that of the N-terminal domain of dihaem FCCs. According to classification based on amino-acid sequence, TpCopC belongs to a high-affinity CopC subfamily characterized by the presence of a conserved His1-Xxx-His3 motif at the N-terminus. Apparently, a unique α-helix which is present in each monomer of TpCopC at the interface with TpFCC plays a key role in complex formation. The structure of the copper-binding site in TpCopC is similar to those in other known CopC structures. His3 is not involved in binding to the copper ion and is 6-7 Å away from this ion. Therefore, the His1-Xxx-His3 motif cannot be considered to be a key factor in the high affinity of CopC for copper(II) ions. It is suggested that the TpFCC-(TpCopC) heterotetramer may be a component of a large periplasmic complex that is responsible for thiocyanate metabolism.
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http://dx.doi.org/10.1107/S2059798318005648DOI Listing
July 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

Identification of branched-chain amino acid aminotransferases active towards (R)-(+)-1-phenylethylamine among PLP fold type IV transaminases.

J Biotechnol 2018 Apr 14;271:26-28. Epub 2018 Feb 14.

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.

New class IV transaminases with activity towards L-Leu, which is typical of branched-chain amino acid aminotransferases (BCAT), and with activity towards (R)-(+)-1-phenylethylamine ((R)-PEA), which is typical of (R)-selective (R)-amine:pyruvate transaminases, were identified by bioinformatics analysis, obtained in recombinant form, and analyzed. The values of catalytic activities in the reaction with L-Leu and (R)-PEA are comparable to those measured for characteristic transaminases with the corresponding specificity. Earlier, (R)-selective class IV transaminases were found to be active, apart from (R)-PEA, only with some other (R)-primary amines and D-amino acids. Sequences encoding new transaminases with mixed type of activity were found by searching for changes in the conserved motifs of sequences of BCAT by different bioinformatics tools.
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http://dx.doi.org/10.1016/j.jbiotec.2018.02.005DOI Listing
April 2018

Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells.

Sci Rep 2017 10 20;7(1):13688. Epub 2017 Oct 20.

Department of Food Sciences and Technology, VIBT - Vienna Institute of BioTechnology, BOKU - University of Natural Resources and Life Sciences, A-1190, Vienna, Austria.

Fungal high redox potential laccases are proposed as cathodic biocatalysts in implantable enzymatic fuel cells to generate high cell voltages. Their application is limited mainly through their acidic pH optimum and chloride inhibition. This work investigates evolutionary and engineering strategies to increase the pH optimum of a chloride-tolerant, high redox potential laccase from the ascomycete Botrytis aclada. The laccase was subjected to two rounds of directed evolution and the clones screened for increased stability and activity at pH 6.5. Beneficial mutation sites were investigated by semi-rational and combinatorial mutagenesis. Fourteen variants were characterised in detail to evaluate changes of the kinetic constants. Mutations increasing thermostability were distributed over the entire structure. Among them, T383I showed a 2.6-fold increased half-life by preventing the loss of the T2 copper through unfolding of a loop. Mutations affecting the pH-dependence cluster around the T1 copper and categorise in three types of altered pH profiles: pH-type I changes the monotonic decreasing pH profile into a bell-shaped profile, pH-type II describes increased specific activity below pH 6.5, and pH-type III increased specific activity above pH 6.5. Specific activities of the best variants were up to 5-fold higher (13 U mg) than BaL WT at pH 7.5.
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http://dx.doi.org/10.1038/s41598-017-13734-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651918PMC
October 2017

Specific titin and myomesin domains stimulate myoblast proliferation.

Biochem Biophys Rep 2017 Mar 30;9:226-231. Epub 2016 Dec 30.

Federal Research Centre of Biotechnology, Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskiy prospect 33, 119071 Moscow, Russia.

Myofibrillar proteins titin and myomesin stimulated myoblast proliferation as determined by MTT-test and labelled thymidine incorporation in the DNA. Specific Fn type III and Ig-like domains of these proteins were able to exert mitogenic effects as well. Proliferative effect of Fn type III domains was highly sensitive to inhibition of Ca/calmodulin dependent protein kinase, whereas the effect of Ig-like domains showed greater sensitivity to the inhibition of adenylyl cyclase - cAMP - PKA pathway. IGF-1 autocrine signalling inhibition partially suppressed mitogenic effects revealed by both domain types.
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http://dx.doi.org/10.1016/j.bbrep.2016.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614584PMC
March 2017

Remembering Navasard V. Karapetyan (1936-2015).

Photosynth Res 2017 Jun 17;132(3):221-226. Epub 2017 Mar 17.

Department of Plant Biology, Department of Biochemistry, and Center of Biophysics & Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.

Navasard Vaganovich Karapetyan (September 6, 1936-March 6, 2015) began his scientific career at the Bach Institute of Biochemistry of the Russian Academy of Sciences, Moscow, and was associated with this institute for over 56 years. He worked in the area of biochemistry and biophysics of photosynthesis and was especially known for his studies on chlorophyll a fluorescence in higher plants and cyanobacteria, molecular organization of Photosystem I, photoprotective energy dissipation, and dynamics of energy migration in the two photosystems. We present here a brief biography and comments on the work of Navasard Karapetyan. We remember him as an enthusiastic person who had an unflagging curiosity, energy and profound sincere interest in many aspects of photosynthesis research.
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http://dx.doi.org/10.1007/s11120-017-0361-3DOI Listing
June 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

Enhanced conformational flexibility of the histone-like (HU) protein from Mycoplasma gallisepticum.

J Biomol Struct Dyn 2018 01 29;36(1):45-53. Epub 2016 Dec 29.

a National Research Centre 'Kurchatov Institute', Kurchatov Complex of NBICS-Technologies , Akad. Kurchatova sqr., 1, Moscow 123182 , Russian Federation.

The histone-like (HU) protein is one of the major nucleoid-associated proteins involved in DNA supercoiling and compaction into bacterial nucleoid as well as in all DNA-dependent transactions. This small positively charged dimeric protein binds DNA in a non-sequence specific manner promoting DNA super-structures. The majority of HU proteins are highly conserved among bacteria; however, HU protein from Mycoplasma gallisepticum (HUMgal) has multiple amino acid substitutions in the most conserved regions, which are believed to contribute to its specificity to DNA targets unusual for canonical HU proteins. In this work, we studied the structural dynamic properties of the HUMgal dimer by NMR spectroscopy and MD simulations. The obtained all-atom model displays compliance with the NMR data and confirms the heterogeneous backbone flexibility of HUMgal. We found that HUMgal, being folded into a dimeric conformation typical for HU proteins, has a labile α-helical body with protruded β-stranded arms forming DNA-binding domain that are highly flexible in the absence of DNA. The amino acid substitutions in conserved regions of the protein are likely to affect the conformational lability of the HUMgal dimer that can be responsible for complex functional behavior of HUMgal in vivo, e.g. facilitating its spatial adaptation to non-canonical DNA-targets.
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http://dx.doi.org/10.1080/07391102.2016.1264893DOI Listing
January 2018

A Novel highly thermostable branched-chain amino acid aminotransferase from the crenarchaeon Vulcanisaeta moutnovskia.

Enzyme Microb Technol 2017 Jan 11;96:127-134. Epub 2016 Oct 11.

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

A new fold-type IV branched-chain amino acid aminotransferase VMUT0738 from the hyperthermophilic Crenarchaeon Vulcanisaeta moutnovskia was successfully expressed in Escherichia coli. Purified VMUT0738 showed activity toward numerous aliphatic and aromatic l-amino acids and 2-oxo acids at optimal pH 8.0. Distinguishing features of the VMUT0738 compared with typical BCAT are the absence of activity toward acidic substrates, high activity toward basic ones, and low but detectable activity toward the (R)-enantiomer of α-methylbenzylamine (0.0076U/mg) The activity of VMUT0738 increases with a rise in the temperature from 60°C to 90°C. VMUT0738 showed high thermostability (after 24h incubation at 70°C the enzyme lost only 27% of the initial activity) and the resistance to organic solvents. The sequence alignment revealed two motifs (V/I)xLDxR and PFG(K/H)YL characteristic of BCATs from species of the related genera Vulcanisaeta, Pyrobaculum and Thermoproteus that might be responsible for the unique substrate recognition profile of the enzyme.
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http://dx.doi.org/10.1016/j.enzmictec.2016.10.002DOI Listing
January 2017
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