ABSTRACT
Recently, we have studied properties and structural features of the thermostable halotolerant alcohol dehydrogenase from archaeon Thermococcus sibiricus (TsAdh319). In the present work, the effect of sodium chloride on activity and thermostability was explored using circular dichroism, fluorescent spectroscopy, and differential scanning calorimetry. The activity of TsAdh319 increased in the presence of NaCl and remained at the elevated level up to 4 M of NaCl. Sodium chloride at molar concentrations reduced the optimal reaction temperature, increased both Michaelis constant (K m) and k cat values for the substrates tested, decreased affinity for the coenzyme, and stoichiometry of coenzyme binding. No changes were revealed in a secondary or quaternary structure of the protein in the presence of NaCl up to 90 °C. According to differential scanning calorimetry, the irreversible unfolding started around 90 °C, the addition of NaCl decreased T m from 104.2 to 102.2 °C, and reduced ΔH from 438 to 348 kJ/mol. Kinetic studies revealed positive effect of NaCl on the TsAdh319 thermostability. The results are interpreted in regard to TsAdh319 structural data.
Subject(s)
Oxidoreductases/chemistry , Oxidoreductases/metabolism , Sodium Chloride/pharmacology , Temperature , Thermococcus/enzymology , Enzyme Stability/drug effects , Protein Denaturation/drug effectsABSTRACT
Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme - half-life of 1 h at 100 °C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 Å resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate + glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous "resilience" of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.
Subject(s)
Alcohol Dehydrogenase/chemistry , Archaeal Proteins/chemistry , Protein Structure, Quaternary , Thermococcus/enzymology , Alcohol Dehydrogenase/genetics , Alcohol Dehydrogenase/metabolism , Amino Acid Sequence , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Hydrophobic and Hydrophilic Interactions , Iron/chemistry , Iron/metabolism , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Models, Molecular , Molecular Sequence Data , NADP/chemistry , NADP/metabolism , Protein Binding , Protein Folding , Protein Multimerization , Protein Structure, Secondary , Sequence Homology, Amino Acid , Substrate Specificity , Temperature , Thermococcus/geneticsABSTRACT
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.
