Ekaterina Yu. Bezsudnova

Characterization of a thermostable short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus.

ABSTRACT Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the hyperthermophilic archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.

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

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.

Experimental and computational studies on the unusual substrate specificity of branched-chain amino acid aminotransferase from Thermoproteus uzoniensis

PLP-Dependent fold-type IV branched-chain amino acid aminotransferases (BCATs) from archaea have so far been poorly characterized. A new BCAT from the hyperthermophilic archaeon Thermoproteus uzoniensis (TUZN1299) has been studied. TUZN1299 was found to be highly active toward branched-chain amino acids (BCAAs), positively charged amino acids, l-methionine, l-threonine, l-homoserine, l-glutamine, as well as toward 2-oxobutyrate and keto analogs of BCAAs, whereas l-glutamate and α-ketoglutarate were not converted in the overall reaction. According to stopped-flow experiments, the enzyme showed the highest specificity to BCAAs and their keto analogs. In order to explain the molecular mechanism of the unusual specificity of TUZN1299, bioinformatic analysis was implemented to identify the subfamily-specific positions in the aminotransferase class IV superfamily of enzymes. The role of the selected residues in binding of various ligands in the active site was further studied using molecular modeling. The results indicate that Glu188 forms a novel binding site for positively charged and polar side-chains of amino acids. Lack of accommodation for α-ketoglutarate and l-glutamate is due to the unique orientation and chemical properties of residues 102-106 in the loop forming the A-pocket. The likely functional roles of TUZN1299 in cellular metabolism - in the synthesis and degradation of BCAAs - are discussed.

Nicotinamidase from the thermophilic archaeon Acidilobus saccharovorans: Structural and functional characteristics

Nicotinamidase is involved in the maintenance of NAD+ homeostasis and in the NAD+ salvage pathway of most prokaryotes, and it is considered as a possible drug target. The gene (ASAC_0847) encoding a hypothetical nicotinamidase has been found in the genome of the thermophilic archaeon Acidilobus saccharovorans. The product of this gene, NA_As0847, has been expressed in Escherichia coli, isolated, and characterized as a Fe2+-containing nicotinamidase (kcat/Km = 427 mM−1·sec−1)/pyrazinamidase (kcat/Km = 331 mM−1·sec−1). NA_As0847 is a homodimer with molecular mass 46.4 kDa. The enzyme has high thermostability (T1/2 (60°C) = 180 min, T1/2 (80°C) = 35 min) and thermophilicity (Topt = 90°C, Ea = 30.2 ± 1.0 kJ/mol) and broad pH interval of activity, with the optimum at pH 7.5. Special features of NA_As084 are the presence of Fe2+ instead of Zn2+ in the active site of the enzyme and inhibition of the enzyme activity by Zn2+ at micromolar concentrations. Analysis of the amino acid sequence revealed a new motif of the metal-binding site (DXHXXXDXXEXXXWXXH) for homological archaeal nicotinamidases.

Structure of the dodecamer of the aminopeptidase APDkam598 from the archaeon Desulfurococcus kamchatkensis.

The crystal structure of the aminopeptidase APDkam589 from the thermophilic crenarchaeon Desulfurococcus kamchatkensis was determined at a resolution of 3.0 Å. In the crystal, the monomer of APDkam589 and its symmetry-related monomers are densely packed to form a 12-subunit complex. Single-particle electron-microscopy analysis confirms that APDkam589 is present as a compact dodecamer in solution. The APDkam589 molecule is built similarly to the molecules of the PhTET peptidases, which have the highest sequence identity to APDkam589 among known structures and were isolated from the more thermostable archaeon Pyrococcus horikoshii. A comparison of the interactions of the subunits in APDkam589 with those in PhTET1, PhTET2 and PhTET3 reveals that APDkam589 has a much lower total number of salt bridges, which correlates with the lower thermostability of APDkam589. The monomer of APDkam589 has six Trp residues, five of which are on the external surface of the dodecamer. A superposition of the structure of APDkam589 with those having a high sequence similarity to APDkam589 reveals that, although the positions of Trp45, Trp252 and Trp358 are not conserved in the sequences, the spatial locations of the Trp residues in these models are similar.

Structures of β-glycosidase from Acidilobus saccharovorans in complexes with tris and glycerol

99 Glycosidases, enzymes catalyzing cleavage of O– glycosidic bonds (EC 3.2.1), are widespread in nature and are promising objects for biotechnologies and cre ation of medicines [1, 2]. The accepted classification of glycosidases based on the homology of amino acid sequences (http://www.cazy.org/) considers more than 110 glycosidase families. The object of this inves tigation is β–glycosidase from the thermoacidophilic archaeon Acidilobus saccharovorans (Asβ Gly, gene ASAC1390 [3]), which belongs to the first glycosidase family (GH1) according to the CAZy classification. Glycosidases GH1 hydrolyze equatorial glycosidic bonds, with the anomeric center configuration being unchanged. The catalytic reaction is performed owing to two glutamate residues; one of them is a proton donor and the other is a nucleophile that attacks the anomeric carbon atom. The basis of the glycosidase GH1 tertiary structure is formed by the (β/α)8 barrel. Among glycosidases with the structures deposited in PDB (http://www.rcsb.org/), the enzymes from Sul folobus solfataricus (Ssβ Glc1, PDB code 1uwq), Thermosphaera aggregans (1qvb), and Pyrococcus furi osus (3apg) are the most homological to Asβ Gly (71, 56, and 54% identities, respectively). In this work, the 3D structures of Asβ Gly in complexes with glycerol and Tris are determined by X ray analysis.

Expression, purification and crystallization of a thermostable short-chain alcohol dehydrogenase from the archaeon Thermococcus sibiricus

Alcohol dehydrogenases belong to the oxidoreductase family and play an important role in a broad range of physiological processes. They catalyze the cofactor-dependent reversible oxidation of alcohols to the corresponding aldehydes or ketones. The NADP-dependent short-chain alcohol dehydrogenase TsAdh319 from the thermophilic archaeon Thermococcus sibiricus was overexpressed, purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion method using 25%(w/v) polyethylene glycol 3350 pH 7.5 as precipitant. The crystals diffracted to 1.68 A resolution and belonged to space group I222, with unit-cell parameters a = 55.63, b = 83.25, c = 120.75 A.

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

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.

Molecular dynamics study of the structural and dynamic characteristics of the polyextremophilic short-chain dehydrogenase from the Thermococcus sibiricus archaeon and its homologues

The study of structural adaptations of proteins from polyextremophilic organisms using computational molecular dynamics method is appealing because the obtained knowledge can be applied to construction of synthetic proteins with high activity and stability in polyextreme media which is useful for many industrial applications. To investigate molecular adaptations to high temperature, we have focused on a superthermostable short-chain dehydrogenase TsAdh319 from the Thermococcus sibiricus polyextremophilic archaeon and its closest structural homologues. Molecular dynamics method is widely used for molecular structure refinement, investigation of biological macromolecules motion, and, consequently, for interpreting the results of certain biophysical experiments. We performed molecular dynamics simulations of the proteins at different temperatures. Comparison of root mean square fluctuations (RMSF) of the atoms in thermophilic alcohol dehydrogenases (ADHs) at 300 K and 358 K revealed the existence of stable r...

NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860: Structural and Functional Features

We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, 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.