K-073. An Iron-Containing Alcohol Dehydrogenase from Hyperthermophilic Archaeon Thermococcus Strain ES1: Heterologous Expression in Escherichia coli and Molecular Characterization of the Recombinant Enzyme

X. Ying1, A. M. Grunden2, L. Nie1, M. W. W. Adams3, K. Ma1;
1Univ. of Waterloo, Waterloo, ON, CANADA, 2North Carolina State Univ., Raleigh, NC, 3Univ. of Georgia, Athens, GA.

The thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was purified and characterized previously. It was necessary to obtain sufficient amount of the active enzyme by heterologous expression for further studying its structural and catalytic characteristics. To achieve this goal, Escherichia coli was used for the over-expression of the gene encoding ES1 ADH. Consequently, the gene was cloned, sequenced and over-expressed in E. coli as desired. The deduced amino acid sequence showed high sequence identity to ADHs from other Thermococcus species (80%), conserved domains and binding motifs for catalytic metal ion and dinucleotide of iron-containing ADHs. Both recombinant and native ES1 ADHs were purified using an FPLC system under anaerobic conditions. The recombinant enzyme was characterized in parallel with the native ES1 ADH. Both enzymes appeared to be homotetramers (175±9 kDa) with a subunit size of 45±1 kDa revealed by SDS-PAGE, which was close to the calculated one from the deduced amino acid sequence (44.8 kDa). The recombinant ADH contained 1±0.1 g-atom iron per subunit determined by inductively coupled plasma mass spectrometry, which was consistent with the value reported for the native enzyme. It was found that both enzymes were sensitive to oxygen, and a half-life upon exposure to oxygen was about 4 min. The recombinant enzyme exhibited a specific activity of 105±2 U/mg, which was very similar to that of the native enzyme (110±3 U/mg). The optimal pHs for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively. Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there was no activity towards methanol and secondary alcohols. Kinetic parameters of both enzymes showed lower Km-values for acetaldehyde and NADPH and higher Km-values for ethanol and NADP+. It is concluded that the gene encoding ES1 ADH was over-expressed successfully in E. coli, and this is the first report that a recombinant version of the iron-containing ADH from hyperthermophiles was fully active.