Effect of Ketosubstrate on the Product Yield in the Transamination Reaction Catalyzed by Transaminase from Thermoproteus uzoniensis.

The study of the equilibrium of reactions catalyzed by thermostable enzymes is in demand for the development of biotechnological enzyme processes. The results of the analysis of equilibrium of transamination reaction catalyzed by thermostable transaminase from the archaeon Thermoproteus uzoniensis are presented below. A comparison of the conversion of substrates was performed for reactions with L-leucine and pyruvate and L-leucine and 2-oxobutyrate at 65°C. The establishment of the equilibrium was controlled by a decrease in the concentration of 2-oxobutyrate or pyruvate and by the accumulation of the keto analog of L-leucine. It was shown that the degree of conversion of L-leucine in the reaction with specific 2-oxobutyrate is higher than in the reaction with nonspecific pyruvate.

Properties of Bacterial and Archaeal Branched-Chain Amino Acid Aminotransferases.

Branched-chain amino acid aminotransferases (BCATs) catalyze reversible stereoselective transamination of branched-chain amino acids (BCAAs) L-leucine, L-isoleucine, and L-valine. BCATs are the key enzymes of BCAA metabolism in all organisms. The catalysis proceeds through the ping-pong mechanism with the assistance of the cofactor pyridoxal 5'-phosphate (PLP). BCATs differ from other (S)-selective transaminases (TAs) in 3D-structure and organization of the PLP-binding domain. Unlike other (S)-selective TAs, BCATs belong to the PLP fold type IV and are characterized by the proton transfer on the re-face of PLP, in contrast to the si-specificity of proton transfer in fold type I (S)-selective TAs. Moreover, BCATs are the only (S)-selective enzymes within fold type IV TAs. Dual substrate recognition in BCATs is implemented via the "lock and key" mechanism without side-chain rearrangements of the active site residues. Another feature of the active site organization in BCATs is the binding of the substrate α-COOH group on the P-side of the active site near the PLP phosphate group. Close localization of two charged groups seems to increase the effectiveness of external aldimine formation in BCAT catalysis. In this review, the structure-function features and the substrate specificity of bacterial and archaeal BCATs are analyzed. These BCATs differ from eukaryotic ones in the wide substrate specificity, optimal temperature, and reactivity toward pyruvate as the second substrate. The prospects of biotechnological application of BCATs in stereoselective synthesis are discussed.

Iron-dependent superoxide dismutase from novel thermoacidophilic crenarchaeon Acidilobus saccharovorans: from gene to active enzyme.

A gene encoding superoxide dismutase was revealed in the genome of the thermoacidophilic crenarchaeon Acidilobus saccharovorans. A recombinant expression vector was constructed and transformed into E. coli cells. The novel recombinant superoxide dismutase was purified and characterized. The enzyme was shown to be an iron-dependent superoxide dismutase able to bind various bivalent metals in the active site. According to differential scanning calorimetric data, the denaturation temperature of the enzyme is 107.3°C. The maximal activity of the Fe(II) reconstituted enzyme defined by xanthine oxidase assay is 1700 U/mg protein. Study of the thermal stability of the superoxide dismutase samples with various metal contents by tryptophan fluorescence indicated that the thermal stability and activity of the enzyme directly depend on the nature of the reconstituted metal and the degree of saturation of binding sites.