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

• Published in: Enzyme and microbial technology 2017-01, Vol.96, p.127-134
• Main Authors: Stekhanova, Tatiana N., Rakitin, Andrey L., Mardanov, Andrey V., Bezsudnova, Ekaterina Yu, Popov, Vladimir O.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Branched-chain amino acid aminotransferase; Cloning, Molecular; Enzyme Stability; Genes, Archaeal; Hydrogen-Ion Concentration; Kinetics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Sequence motifs; Structural Homology, Protein; Substrate Specificity; Temperature; Thermoproteaceae - enzymology; Thermoproteaceae - genetics; Thermostability; Transaminases - genetics; Transaminases - metabolism
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2016.10.002

•A novel archaeal BCAT was expressed in E. coli, purified and characterized.•Enzyme showed a broad spectrum and unique combination of substrate specificities.•VMUT0738 showed high (S)-enantioselectivity, thermostability, resistance to solvents.•Two sequence motifs characteristic of BCATs from Thermoproteaceae were revealed. 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.