Annotation of the results obtained in 2018
As part of our continuing research on main proteins involved in energy metabolism of haloalkaliphilic bacteria of the genus Thioalkalivibrio, the structure and functions of the new copper-dependent enzyme thiocyanate dehydrogenase (TCDH) from the bacterium Tv. paradoxus were further explored. According to our data, the structure of the catalytic copper cluster of TcDH composed of three copper ions is unique among copper-containing proteins, and this structure mediates catalysis of the previously unknown oxidation of thiocyanate to cyanate and elemental sulfur. In order to elucidate the molecular mechanism of action of TcDH, the following studies were performed: - the procedure for activation of TcDH by copper ions was optimized with monitoring enzyme activity and metal content by ICP-MS and EPR. The results of investigation confirmed the fact that TcDH exhibits the highest activity upon binding of three copper ions per protein subunit. -the mutant of TcDH, in which the residue His482 coordinating the copper ion Cu3 is replaced by Gln, was functionally and structurally characterized. An amino acid sequence analysis of homologous of TcDH demonstrated that His482 is the least conserved catalytically important residue. According to the structural data, the His482Gln mutant of TcDH binds two copper ions. The copper ion Cu3 is absent in the mutant structure. The H482Q mutant of TcDH containing two copper ions in the active site does not display catalytic activity in the oxidation reaction of thiocyanate, which supports our hypothesis that at least three copper ions are required for the oxidation of thiocyanate. Besides, these results cast doubt on the fact that homologous of TcDH under consideration possess thiocyanate dehydrogenase activity. - monoheme cytochrome c (С552, WP_006748979.1) with a molecular weight of 18 kDa was identified and characterized as a potential electron acceptor for TcDH in the oxidation of thiocyanate in the cell. In the genomes of bacteria of the genus Thioalkalivibrio, the С552 gene is located near the operon containing the gene encoding TcDH. Recombinant С552 was produced. It was shown that С552 can serve as an electron acceptor in the TcDH-catalyzed in vitro oxidation of thiocyanate. The effect of рН and the ionic strength on the rate of the reaction with С552 as an electron acceptor was characterized. The potential of the heme c in C552 was estimated by potentiometric titration at 130 mV. The results of the structural and functional studies of wild-type TcDH and its mutants obtained during the implementation of this project are not in contradiction to the molecular mechanism of catalytic action of TcDH, which we have proposed previously based on molecular dynamics simulations and quantum chemical calculations. In order to elucidate the mechanisms of adaptation of enzymes from haloalkaliphilic bacteria of the genus Thioalkalivibrio, we performed a comparative analysis of the structures of homologous flavocytochrome c dehydrogenases (FCCs) from the haloalkaliphilic bacterium Tv. paradoxus, the mesophilic neutrophilic bacterium Allochromatium vinosum, and the thermophilic bacterium Thermochromatium tepidum. An analysis revealed a decrease in the percentage of lysines involved in the formation of solvent-accessible surface of FCC from the haloalkaliphilic bacterium compared to FCCs from two other organisms. This structural feature is characteristic of the mechanisms of adaptation of both halo- and alkaliphilic proteins and is associated with high hydrophobicity of the aliphatic side chain of lysine, which is uncharged at alkaline pH values and facilitates aggregation of molecules in high-salinity solutions. Earlier, we have noted this distinguishing feature as one of mechanisms of adaptation of octaheme nitrite reductases from the bacteria Tv. paradoxus (Popinako A et al., 2017, PLoS One; 12(5):e0177392). Previously, we have proposed the mechanism of structural adaptation of octaheme nitrite reductases based on the comparative analysis of the structures of enzymes from haloalkaliphilic bacteria of the genus Thioalkalivibrio and the three-dimensional models of homologous enzymes from neutrophilic non-halophilic bacteria of the genus Geobacter. To verify this mechanism, we determined the three-dimensional structure of octaheme nitrite reductase from the bacterium Geobacter ammonificans (GaNiR) and performed the preliminary comparative analysis of this structure and the structures of nitrite reductases from haloalkaliphilic bacteria of the genus Thioalkalivibrio (TvNiR). Significant changes were found in the structures of the monomers in the contact area between two trimers in the TvNiR structure. The three-dimensional arrangement of additional loops in the structure of the monomer of GaNiR interferes with the formation of a hexameric structure of this enzyme. Previously, we have demonstrated that the formation of a hexamer is one of the main mechanisms of stabilization of TvNiR in high-salinity solutions, which correspond to conditions of functioning of TvNiR in the periplasm of haloalkaliphilic bacteria. The structural and functional characterization of new pyridoxal 5’-phosphate (PLP)-dependent class III transaminases (PLP fold type I) and class IV transaminases (PLP fold type IV), which are stereospecific in reactions with primary (R)- and (S)-amines were continued. These enzymes are of interest for biotechnological applications. Our studies performed in 2018 were focused on investigation of biotechnologically important properties of the enzymes, such as stability at different temperatures and in organic solvents, and characterization of transamination products. The structural basis of substrate specificity of the new transaminases was studied in detail. Stability of PLP-dependent fold type I transaminase from the cold-active bacterium Psychrobacter cryohalolentis (Pcryo361) was analyzed. Upon incubation in a 25% (v/v) organic solvent for 24 h at 35 °С, the stability of the enzyme (1–3 mg/mL) was shown to decrease in the order DMSO>DMFA>methanol>acetonitrile. Dimethyl sulfoxide proved to be the solvent of choice. Thus, Pcryo361 retains 60% of activity after incubation in 50% DMSO at 35 °С. Up to 25% of DMSO can be added directly to the reaction mixture in order to increase the solubility of substrates/products. The distinguishing features of Pcryo361 is its unique activity in the amination of alpha-diketones with (S)-alpha-methylbenzylamine as the amino group donor at 10–35 °С. The kinetic parameters of amination of diketones at different temperatures were determined, and it was demonstrated that the specificity of Pcryo361 toward butane-2,3-dione remains unchanged in the reaction temperature range of 10–35 °С. Moreover, the enzyme is characterized by low-temperature activity with amines, diketones, and aldehydes and is resistant to denaturation at 0–10 °С. 2,3,5,6-Tetramethylpyrazine was identified by GC-MS as the amination product of butane-2,3-dione. The amination of pentane-2,3-dione affords heterocyclic aromatic compounds, such as 2,6-diethyl-3,5-dimethylpyrazine and/or 2,5-diethyl-3,6-dimethylpyrazine. Apparently, these compounds are produced via dimerization followed by oxidative aromatization of the initial amination products (ketoamines). Substituted pyrazines are employed as cosmetic, flavor, and fragrance agents. According to the amino acid sequence and characteristic motifs involved in the active site, Pcryo361 belongs to DAPA transaminases (7,8-diaminopelargonic acid transaminases, EC 2.6.1.62) with narrow specificity toward S-adenosyl-L-methionine (SAM) and 7,8-ketoaminopelargonic acid (KAPA). The structural analysis and molecular modeling showed that the active site of Pcryo361 possesses substrate-specific motifs characteristic of both DAPA transaminases and S-amine transaminases. It was shown that specificity of Pcryo361 toward the non-typical substrate, viz., the aromatic amine S-alpha-methylbenzylamine, is related to specificity of the enzyme toward SAM and KAPA. The binding of the amine is observed in the recognition region of the hydrophobic parts of the substrates SAM and KAPA in the active site of the enzyme and is enhanced due to overall mobility of the residues in the active site. The detailed structural analysis of PLP-dependent fold type IV transaminases from the thermophilic bacterium Thermobaculum terrenum (ТА-ТТ) and the halotolerant bacterium Haliangium ochraceum (Hoch3033) was carried out. Both transaminases have substitutions in the characteristic motifs responsible for substrate specificity of PLP fold type IV transaminases. The active site arrangement of Hoch3033 was shown to be similar to that of branched-chain amino acid transaminases (BCATs). However, the active site of Hoch3033 has some differences from the ВСАТ active site: (1) the binding site of the negatively charged carboxyl group in the large pocket is absent, (2) the large pocket is more hydrophobic compared to typical ВСАТs; (3) of the two binding sites of the alpha-carboxyl group of alpha-amino acids as substrates, only one site is present in the small pocket. These features lead to a change in the substrate specificity profile of Hoch3033 compared to typical ВСАТs. Thus, this enzyme is inactive with alpha-ketoglutarate and pyruvate, is active with the aromatic amine (R)-alpha-methylbenzylamine, and catalyzes the unique amination reaction of keto analogues of branched amino acids with (R)-alpha-methylbenzylamine as the amino group donor giving L-amino acid and acetophenone. Mutants of TA-TT were further functionally characterized in order to increase the efficiency of binding of primary R-amines and elucidate the role of residues involved in the active site of ТА-ТТ. The G41V + Y101F (М10) mutant with substitutions in the small pocket and the G41V+Y101F + Y166W+F39Y (М11) and R43S+G41V+Y101F+R115+ Y166W+F39Y (М12) mutants with substitutions in the small and large pockets were constructed and characterized. The double mutation Y166W+F39Y (the replacements were proposed based on the comparison of the active sites of ТА-ТТ and specific (R)-amine transaminases) did not lead to an increase in the catalytic efficiency of ТА-ТТ in the reaction with (R)-alpha-methylbenzylamine. Apparently, the substitutions in the small pocket are most favorable for enhancement of catalytic efficiency of ТА-ТТ in the reaction with primary (R)-amines with retention of its thermal stability. During the project implementation, the structure of the R43S+G41V+Y101F mutant of ТА-ТТ with three substitutions in the small pocket of the active site was determined at 1.85 Å resolution and analyzed. Three substitutions did not change the main chain folding in the small pocket of the active site, but led to a substantial increase in the hydrophobicity of the small pocket, which apparently resulted in an increase in the affinity of the mutant for the aromatic amine (R)-alpha-methylbenzylamine and, as a consequence, in the enhancement of catalytic efficiency of ТА-ТТ in the corresponding half-reaction. The studies within the framework of the project were implemented in full in 2018. Ten articles were published, six of which were in journals belonging to the first quartile (Q1).

 

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Annotation of the results obtained in 2017
The goal of this project is to characterize new enzymes and enzyme systems from extremophilic microorganisms, investigate the mechanisms of catalysis by enzymes with unusual functions, and analyze their three-dimensional structures in order to reveal characteristic features of the structural organization of enzymes from extremophilic microorganisms responsible for their stability and efficient functioning in the habitat conditions. Within the framework of investigation of main enzymes of energy metabolism from haloalkaliphilic bacteria of the genus Thioalkalivibrio, the structure and functions of the new copper-dependent enzyme thiocyanate dehydrogenase (TCDH) from the bacterium Tv. paradoxus were further explored. The main purpose of this stage of the project was to elucidate and validate the mechanism of action of TcDH based on the results of structural and functional analysis, site-directed mutagenesis, EPR spectroscopy, and QM/MM simulations. In order to achieve reproducible results and perform accurate comparison of different TcDH preparations, the procedure for the activation of wild-type and recombinant TcDH with copper ions was standardized. Fully active TcDH preparations were shown to contain at least three copper ions. The EPR studies demonstrated that, regardless of the activation procedure, all copper ions are in the oxidized state. The structure of recombinant TcDH containing three copper ions in the active site in complex with a iodide ion as the inhibitor was determined (2.0 Å resolution). This structure was used for QM/MM simulations of the molecular events in thiocyanate decomposition at the active site of TcDH. In the final structural model of the enzyme–substrate complex, the sulfur atom of thiocyanate is located between type 2 and 3 copper ions, like the iodide ion in the starting structure, and it forms a coordination bond with the type 3 copper ion, while the nitrogen atom of thiocyanate forms a coordination bond with the type 1 copper ion. The conserved water molecule W1, which is present in both the experimental and theoretical structures, is involved in the hydrogen bonding with the residues Glu288 and His136. The abstraction of a proton from the water molecule W1 and its transfer to His136 gives rise to catalytic hydroxyl coordinated to the type 2 copper ion. This hydroxyl attacks the carbon atom of thiocyanate to form a transition state. Based on QM/MM simulations, the putative mechanism of the chemical step of the enzymatic reaction involves the one-step C-S bond cleavage with an activation energy of 16.4 kcal/mol. According to the transition state theory, this value corresponds to the rate constant of 9 s–1 at 303 K, which is higher than the experimental rate constant for the reaction with cytochrome c (5.4 s–1) and is similar to the rate constant for the reaction with low-molecular-weight electron acceptors (8–13 s–1). Apparently, the electron transfer to the acceptor rather than the C–S bond cleavage is the rate-determining step in the reaction with cytochrome c. Mutations of the catalytically important residues His136 and Glu288 gave rise to inactive preparations. This fact confirms the role of these residues in catalysis and the putative mechanism of catalysis. The mutation of the residue His482, which is involved in coordination to the type 3 copper ion, by Gln also resulted in the inactive enzyme. Crystals of the His482Gln mutant suitable for X-ray diffraction were grown in order to reveal the structural factors responsible for inactivation of TcDH due to this mutation. Studies were continued on homologous octaheme nitrite reductases from haloalkaliphilic bacteria of the genus Thioalkalivibrio and neutrophilic non-haloalkaliphilic bacteria of the genus Geobacter. In this project, the proteins were analyzed in relation to the structural mechanisms of adaptation of the proteins to extreme conditions. In order to perform a comparative analysis of the enzyme structures, octaheme nitrite reductase (GaNiR) from Geobacter ammonificans was isolated and characterized for the first time. Crystals of GaNiR were grown, and the X-ray diffraction data set was collected from these crystals to 1.8 Å resolution. Besides, this enzyme is of interest because it is the first octaheme nitrite reductase, which was demonstrated to be involved in respiratory ammonification of nitrite in the cell. Earlier, octaheme nitrite reductase (TvNiR) from the haloalkaliphilic bacterium Tv. nitratireducens was isolated in our laboratory; however, the function of this protein in the cell was not quite clear. Studies on the structural and functional characterization of new class III and IV transaminases stereospecific for (R)- and (S)-primary amines were continued. These proteins are of great interest for biotechnology. In 2017, the biotechnological part of the project was focused on the analysis of stability and substrate specificity of transaminases and the consideration of their structures in order to elucidate specific features of the active site organization. Stability of transaminases (TA) from the bacterium Thermobaculum terrenum and the archaea Methanococcus vanielii in organic solvents was analyzed. It was found that TA from T. terrenum retains 100% activity after 24 h incubation at 50 °С in 50% dimethyl sulfoxide (DMSO) and 50% acetonitrile; 70 and 85% activity is retained in 50% methanol and 50% ethanol, respectively, in Tris buffer, рН 8.0. Besides, the addition of 15% of the solvent (DMSO, acetonitrile, or methanol) to the reaction mixture at 50 °С was shown to lead to a 1.5–1.6-fold increase in TA activity. These results provide evidence that TA from T. terrenum is stable in aqueous organic mixtures. The operational stability of the enzyme allows the addition of organic solvents to the reaction mixture. Unexpectedly, TA from the archaea M. vanielii proved to be unstable at high temperatures and in aqueous organic mixtures. Therefore, this TA will not be considered in further studies as an enzyme promising for biothechnology. A comparison of the properties of two transaminases confirms the fact that thermal stability of the enzymes correlates with stability in aqueous organic media. Within the framework of the project, the structures of the holo form of TA from T. terrenum, the holo form of TA from M. Vanielii, and the complex of the latter enzyme with the irreversible inhibitor gabaculine were analyzed. A comparative analysis of the substrate-binding cavity and the active site of both transaminases was performed. The small substrate-binding pocket in TA from M. vanielii was found to be smaller than that in TA from T. terrenum, whereas the large pockets are similar in size but are substantially different in shape due to the different arrangement of large hydrophobic groups involved in the formation of this pocket. These differences apparently affect the substrate specificity of the enzymes. Detailed analysis of the residues involved in the formation of the large and small pockets of the substrate-binding cavity was performed for TA from T. terrenum. The structural elements and individual residues, which are, in our opinion, responsible for a new type of double substrate recognition revealed for TA from T.terrenum, were indentified. Thus, this enzyme recognizes the negatively charged keto substrate (alpha-ketoglutarate) and the amine substrate (aromatic R-primary amine) at a single active site. It was demonstrated that both charged groups and hydrophobic moieties of substrate molecules can be bound in the large and small pockets of the active site of TA from T. terrenum. The binding of hydrophobic aromatic amine was refined by the molecular docking of (R)-alpha-methylbenzylamine (R-MBA) to the active site of the transaminase. In order to enhance the efficiency of TA from T.terrenum in the reaction with (R)-primary amines, mutants at the residues involved in the small pocket (M1:R43S, M2:R43S+Y101F, M3:R43S+G41V+Y101F) , the large pocket (M4:Arg115), and both in the large and small pockets (М5: R43S+G41V+Y101F+F39Y, M6: R43S+G41V+Y101F+W32H, M7: R43S+G41V+Y101F+F39Y+W32H, M8: R43S+G41V+Y101F+S115R, M9: R43S+G41V+Y101F+S115R+insertion А108) were constructed and isolated. The choice of mutations was determined by the conserved nature of a particular residue and its role in the catalysis by fungal R-amine:pyruvate transaminases. The mutation of Arg43 in the small pocket led to a decrease in the activity of TA from T.terrenum in the overall reaction due to violation of coordination of the alpha-carboxyl group of the keto substrate (pyruvate or alpha-ketoglutarate). The mutant M3 proved to be totally inactive in the overall reaction. However, the kinetic parameters of the half-reaction with the substrate R-MBA indicate that the efficiency of deamination of R-MBA by M3 is 21 times higher. The mutant М8 proved to be most efficient in the half-reaction with R-MBA. Thus, the efficiency of deamination of R-MBA was increased by two orders of magnitude. It should be noted that the S115R mutation led to partial restoration of the enzyme activity in the overall reaction with the keto substrate alpha-ketoglutarate. Therefore, a new alpha-keto substrate-binding site appears in the enzyme instead of the destroyed substrate-binding site in the small pocket. The pH optimum of the overall reaction (R-MBA+ alpha-ketoglutarate) shifted to рН 7.0. The single S115R mutation in the wild-type enzyme (mutant М4) did not lead to a considerable enhancement of the efficiency of deamination of R-MBA; besides, the activity of М4 in the overall reaction decreased to that of М8. An elongation of the loop (insertion A108 in M9) did not lead to the improvement of the R-MBA binding and an increase in the activity of the enzyme in the overall reaction. The mutants М5, М6, and М7 were inactive in the overall reaction. The efficiency of deamination of R-MBA by these mutants is higher compared to the wild-type enzyme but lower than that for М3. Therefore, the mutagenesis resulted in the enhancement of the efficiency of deamination of R-MBA, but the rate of the overall transamination reaction catalyzed by TA from T. terrenum has not yet increased. Studies on the structural and functional characterization of omega-transaminase from the psychrophilic bacterium Psychrobacter cryohalolentis (Pcryo0361) were continued. This transaminase belongs to a poorly studied family of 7,8-diaminopelargonic acid transaminases (DAPA_TA) with narrow specificity and is characterized by specificity for (S)-alpha-methylbenzylamine, aldehydes, and diketones, which is not typical of DAPA_TA. The specificity of the enzyme for diketones and the low-temperature enzyme activity were characterized in detail. It was found that the specific activity of Pcryo0361 in the overall reaction at 35 ºС decreases in the series benzaldehyde>2,3-hexanedione>2,3-pentadione>2,3-butanedione>isobutanal. The specific activity toward diketones at 35 °С is 0.17–0.24 U/mg (this is a high level of activity for this family of transaminases, even toward the specific substrate 7-keto-8-aminopelargonic acid). At 0 °С, Pcryo0361 retains 70% activity (with respect to activity at 35 °С) in the overall reaction with 2,3-pentanedione. The activity of Pcryo0361 in the standard experiment remains unchanged for 4 h after the incubation at 0 ºС in 100 mM carbonate buffer, pH 10.0. The incubation at 5 ºС under the same conditions led to a 30% decrease in the enzyme activity in the standard experiment during the first hour and then the activity remained unchanged for 3 h. In other words, cold denaturation was not observed during this period of time and, in addition, the enzyme retained activity, as opposed to incubation at 35 °С, for several hours. The refined structure of Pcryo0361 was deposited in the Protein Data Bank RCSB (www.rcsb.org); PDB ID 6ERK.

 

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