Cloning and over-expression in Escherichia coli of the gene encoding NADPH group III alcohol dehydrogenase from Thermococcus hydrothermalis. Characterization and comparison of the native and the recombinant enzymes.
A NADP-dependent group III alcohol dehydrogenase (ADH) was purified from the hyperthermophilic strictly anaerobic archaeon Thermococcus hydrothermalis, which grows at an optimum temperature of 85 degrees C and an optimum pH of 6. The gene encoding this enzyme was cloned, sequenced, and over-expressed in Escherichia coli. The recombinant enzyme was purified, characterized and compared with the native form of the enzyme. The enzyme structure is pH-dependent, being a 197-kDa tetramer (subunit of 45 kDa) at pH 10.5, the pH optimum for alcohol oxidation, and a 80.5-kDa dimer at pH 7.5, the pH optimum for aldehyde reduction. The kinetic parameters of the enzyme show that the affinity of the enzyme is greater for the aldehyde substrate and NADPH cofactor, suggesting that the dimeric form of the enzyme is probably the active form in vivo. The ADH of T. hydrothermalis oxidizes a series of primary aliphatic and aromatic alcohols preferentially from C2 to C8 but is also active towards methanol and glycerol and stereospecific for monoterpenes. T. hydrothermalis ADH is the first Thermococcale ADH to be cloned and overproduced in a mesophilic heterologous expression system, and the recombinant and the native forms have identical main characteristics.
Antoine
E
E
Laboratoire de Biotechnologie des Microorganisms Hydrothermaux, Centre IFREMER de Brest, France. eantoine@ifremer.fr
Rolland
J L
JL
Raffin
J P
JP
Dietrich
J
J
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D019641:DNA, Archaeal / Q000235:genetics; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D019766:Genes, Archaeal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics*
D019641:DNA, Archaeal; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D009249:NADP; D000426:Alcohol Dehydrogenase