Counterbalance of Stability and Activity Observed for Thermostable Transaminase from Thermobaculum terrenum in the Presence of Organic Solvents

Pyridoxal-5’-phosphate-dependent transaminases catalyze stereoselective amination of organic compounds and are highly important for industrial applications. Catalysis by transaminases often requires organic solvents to increase the solubility of reactants. However, natural transaminases are prone to inactivation in the presence of water-miscible organic solvents. Here, we present the solvent tolerant thermostable transaminase from Thermobaculum terrenum (TaTT) that catalyzes transamination between L-leucine and alpha-ketoglutarate with an optimum at 75 °C and increases the activity ~1.8-fold upon addition of 15% dimethyl sulfoxide or 15% methanol at high but suboptimal temperature, 50 °C. The enhancement of the activity correlates with a decrease in the thermal denaturation midpoint temperature. The blue-shift of tryptophan fluorescence suggested that solvent molecules penetrate the hydration shell of the enzyme. Analysis of hydrogen bonds in the TaTT dimer revealed a high number of salt bridges and surface hydrogen bonds formed by backbone atoms. The latter are sensitive to the presence of organic solvents; they rearrange, conferring the relaxation of some constraints inherent to a thermostable enzyme at low temperatures. Our data support the idea that the counterbalance of stability and activity is crucial for the catalysis under given conditions; the obtained results may be useful for fine-tuning biocatalyst efficiency

Overexpression, purification and crystallization of a thermostable DNA ligase from the archaeon Thermococcus sp. 1519

Recombinant ATP-dependent DNA ligase from the archaeon Thermococcus sp. 1519 was overexpressed, purified and used to produce crystals that diffracted to 3.09 Å resolution

Overexpression, purification and crystallization of a thermostable DNA ligase from the archaeon Thermococcus sp. 1519.

DNA ligases catalyze the sealing of 5'-phosphate and 3'-hydroxyl termini at single-strand breaks in double-stranded DNA and their function is essential to maintain the integrity of the genome in DNA metabolism. An ATP-dependent DNA ligase from the archaeon Thermococcus sp. 1519 was overexpressed, purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion method employing 35%(v/v) Tacsimate pH 7.0 as a precipitant and diffracted X-rays to 3.09 A resolution. They belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 79.7, c = 182.6 A

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

The expression, purification and crystallization of a thermostable short-chain alcohol dehydrogenase from the archaeon T. sibiricus is reported