Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1.
The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both enzymes appeared to be homotetramers with a subunit size of 45+/-1 kDa as revealed by SDS-PAGE, which was close to the calculated value (44.8 kDa). The recombinant ADH contained 1.0+/-0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105+/-2 U mg(-1), which was very similar to that of the native enzyme (110+/-3 U mg(-1)). The optimal pH-values for both enzymes 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 the enzymes showed lower K (m)-values for acetaldehyde and NADPH and higher K (m)-values for ethanol and NADP(+). It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile.
Ying
Xiangxian
X
Department of Biology, University of Waterloo, 200 University Ave. W, Waterloo, ON N2L 3G1, Canada.
Grunden
Amy M
AM
Nie
Lin
L
Adams
Michael W W
MW
Ma
Kesen
K
D000426:Alcohol Dehydrogenase / Q000737:chemistry*; D000595:Amino Acid Sequence; D001105:Archaea; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D019712:Thermococcus / Q000378:metabolism*
D011994:Recombinant Proteins; D009249:NADP; D007501:Iron; D000426:Alcohol Dehydrogenase