Sodium Chloride-Induced Modulation of the Activity and Thermal Stability of Short-Chain Oxidoreductase from the Archaeon Thermococcus sibiricus

Author： Stekhanova Tatiana Bezsudnova Ekaterina Mardanov Andrey Gumerov Vadim Artemova Natalya Kleymenov Serguey Popov Vladimir

Publisher： Humana Press, Inc

ISSN： 0273-2289

Source： Applied Biochemistry and Biotechnology, Vol.171, Iss.7, 2013-12, pp. : 1877-1889

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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 ∆H 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.