Cloning and expression of the gene encoding the Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase and biochemical characterization of the enzyme.
The adhB gene encoding Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (S-ADH) was cloned, sequenced and expressed in Escherichia coli. The 1056 bp gene encodes a homotetrameric recombinant enzyme consisting of 37.7 kDa subunits. The purified recombinant enzyme is optimally active above 90 degrees C with a half-life of approx. 1.7 h at 90 degrees C. An NADP(H)-dependent enzyme, the recombinant S-ADH has 1400-fold greater catalytic efficiency in propan-2-ol oxidation than in ethanol oxidation. The enzyme was inactivated by chemical modification with dithionitrobenzoate (DTNB) and diethylpyrocarbonate, indicating that Cys and His residues are involved in catalysis. Zinc was the only metal enhancing S-ADH reactivation after DTNB modification, implicating the involvement of bound zinc in catalysis. Arrhenius plots for the oxidation of propan-2-ol by the native and recombinant S-ADHs were linear from 25 to 90 degrees C when the enzymes were incubated at 55 degrees C before assay. Discontinuities in the Arrhenius plots for propan-2-ol and ethanol oxidations were observed, however, when the enzymes were preincubated at 0 or 25 degrees C. The observed Arrhenius discontinuity therefore resulted from a temperature-dependent, catalytically significant S-ADH structural change. Hydrophobic cluster analysis comparisons of both mesophilic and thermophilic S-ADH and primary- versus S-ADH amino acid sequences were performed. These comparisons predicted that specific proline residues might contribute to S-ADH thermostability and thermophilicity, and that the catalytic Zn ligands are different in primary-alcohol dehydrogenases (two Cys and a His) and S-ADHs (Cys, His, and Asp).
Burdette
D S
DS
Biochemistry Department, Michigan State University, East Lansing 48824, USA.
Vieille
C
C
Zeikus
J G
JG
D000426:Alcohol Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli; D005798:Genes, Bacterial; D016988:Gram-Positive Asporogenous Rods, Irregular / Q000201:enzymology* / Q000235:genetics*; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013816:Thermodynamics
D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase