Abstract
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.
Highlights
Pyridoxal-5’-phosphate (PLP)-dependent transaminases (TAs) catalyze stereoselective amination of keto acids, ketones and aldehydes and are of particular interest as industrially relevant enzymes.Catalysis by TAs requires the application of organic solvents to increase the solubility of reactants and drive the reaction equilibrium towards the amination of ketones [1]
We examined the effects of 15% methanol and 15% dimethyl sulfoxide (DMSO) on transaminase from Thermobaculum terrenum (TaTT) activity in the transamination and methanol
We examined the effects of 15% methanol and 15% DMSO on TaTT activity in the transamination reaction between L-Leu and -ketoglutarate at different temperatures
Summary
Pyridoxal-5’-phosphate (PLP)-dependent transaminases (TAs) catalyze stereoselective amination of keto acids, ketones and aldehydes and are of particular interest as industrially relevant enzymes. It is considered that water-miscible organic solvent–water mixtures destabilize the protein globule: solvent molecules penetrate the hydration shell of the enzyme molecules and strip water from the surface, solvating hydrophobic patches and disrupting the surface hydrogen bonds due to the interaction with backbone atoms [7,22,23,24,25,26]. We focus on the enhancement of the activity of recombinant thermostable transaminase from Thermobaculum terrenum (TaTT) in water–methanol and water–DMSO media Both DMSO and methanol are water-miscible organic solvents and are employed in biocatalytic applications.
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