A new alcohol dehydrogenase, reactive towards methanol, from Bacillus stearothermophilus.
An NAD+-dependent alcohol dehydrogenase (ADH) was purified to homogeneity from an aerobic strain of Bacillus stearothermophilus, DSM 2334 (ADH 2334), and compared with the ADH from B. stearothermophilus NCA 1503 (ADH 1503). When an antibody raised against ADH 2334 was used, no cross-reactivity with ADH 1503 was observed on Western blots; by means of an enzyme-linked-immunoabsorbent-assay ('e.l.i.s.a.') procedure, it was found that ADH 1503 had less than 6% of the antigenic activity of ADH 2334. Amino acid analyses detected very small differences in composition, equivalent to about 40 sequence changes, between the two enzymes. The new enzyme has the same six-amino-acid N-terminal sequence as ADH 1503. ADH 2334, but not ADH 1503, is reactive towards methanol; both enzymes can oxidize ethanol, propan-1-ol, butan-1-ol and butan-2-ol. The new enzyme has a distinctive pH optimum at pH 5.5-6 and has significantly lower KEthanolm and kEthanolcat. values than those of ADH 1503. From steady-state kinetic parameters of the reaction with ethanol, propan-1-ol and butan-1-ol, it was shown that ADH 2334 has an ordered mechanism in both directions, with NAD+ being the compulsory first substrate in alcohol oxidation and NADH release being the rate-limiting step. ADH 1503 has an ordered addition of NAD+ and alcohol, but NADH release is not rate-limiting.
Department of Biochemistry, Trinity College, Dublin, Ireland.
M C
MC
Sheehan
C J
CJ
Bailey
B C
BC
Dowds
D J
DJ
McConnell
D000426:Alcohol Dehydrogenase / Q000276:immunology / Q000378:metabolism*; D000438:Alcohols / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D000432:Methanol / Q000378:metabolism*
D000438:Alcohols; D000596:Amino Acids; D007527:Isoenzymes; D000426:Alcohol Dehydrogenase; D000432:Methanol