Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species.In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beyerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. bejerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native Mr of between 90,000 and 100,000 and a subunit Mr of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NADW-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kc,./Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, T7hermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants. Alcohol dehydrogenase (ADH) is widespread in nature.The ADHs of human and horse liver and baker's yeast are well characterized (7,15,30). Although the metabolism of ethanol is a recognized physiological role of ADH, isozymes of ADH often show higher levels of activity toward other substrates. ADHs react with normal and branched-chain aliphatic and aromatic alcohols, both primary and secondary, as well as the corresponding aldehydes and ketones. The broad substrate range has led to suggestions for other roles for ADH (42), and it remains difficult to determine the physiological reaction and significance of an ADH when multiple ADHs are present in an organism. In anaerobic bacteria that produce alcohols as major end products (27) or that grow on alcohols (e.g., references 5 and 16), the physiological role of the predominant ADH is the formation or oxidation of the pertinent alcohols.Although ADHs generally have a broad substrate range, few are equally reactive toward both primary and secondary alcohols. For example, the liver ADHs react with both primary and secondary alcohols (12). Among the liver ADHs, the human aa isozyme (class I) is far more efficient than the others in oxidizing secondary alcohols (49). Nevertheless, the human wx ADH is still considered a primary ADH because it reacts with primary alcohols more efficiently than with sec...

The enzyme 3-hydroxybutyryl-coenzyme A (CoA) dehydrogenase has been purified 45-fold to apparent homogeneity from the solvent-producing anaerobe Clostridium beierinckii NRRL B593. The identities of 34 of the N-terminal 35 amino acid residues have been determined. The enzyme exhibited a native Mr of 213,000 and a subunit Mr of 30,800. It is specific for the (S)-enantiomer of 3-hydroxybutyryl-CoA. Michaelis constants for NADH and acetoacetyl-CoA were 8.6 and 14 pM, respectively. The maximum velocity of the enzyme was 540 FImol min-1 mg-1 for the reduction of acetoacetyl-CoA with NADH. The enzyme could use either NAD(H) or NADP(H) as a cosubstrate; however, kC,dKm for the NADH-linked reaction was much higher than the apparent value for the NADPH-linked reaction. Also, NAD(H)-linked activity was less sensitive to changes in pH than NADP(H)-linked activity was. In the presence of 9.5 FM NADH, the enzyme was inhibited by acetoacetyl-CoA at concentrations as low as 20 ,uM, but the inhibition was relieved as the concentration of NADH was increased, suggesting a possible mechanism for modulating the energy efficiency during growth.