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A new alcohol dehydrogenase, reactive towards methanol, from Bacillus stearothermophilus.

ABSTRACT: 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.

SUBMITTER: Sheehan MC

PROVIDER: S-EPMC1149199 | biostudies-other | 1988 Jun

REPOSITORIES: biostudies-other

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Project description:The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

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A new alcohol dehydrogenase, reactive towards methanol, from Bacillus stearothermophilus.

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