Publications

• Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii.

  Ismaiel A.A., Zhu C.X., Colby G.D., Chen J.S.

  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 beijerinckii. An alcohol dehydrogenase (ADH ... >> More

  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 beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. 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 M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-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 kcat/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, Thermoanaerobium 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. << Less

  J. Bacteriol. 175:5097-5105(1993) [PubMed] [EuropePMC]

• Biochemical and structural properties of chimeras constructed by exchange of cofactor-binding domains in alcohol dehydrogenases from thermophilic and mesophilic microorganisms.

  Goihberg E., Peretz M., Tel-Or S., Dym O., Shimon L., Frolow F., Burstein Y.

  The cofactor-binding domains (residues 153-295) of the alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii (TbADH), the mesophilic bacterium Clostridium beijerinckii (CbADH), and the protozoan parasite Entamoeba histolytica (EhADH1) have been exchanged. Three chimeras have been ... >> More

  The cofactor-binding domains (residues 153-295) of the alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii (TbADH), the mesophilic bacterium Clostridium beijerinckii (CbADH), and the protozoan parasite Entamoeba histolytica (EhADH1) have been exchanged. Three chimeras have been constructed. In the first chimera, the cofactor-binding domain of thermophilic TbADH was replaced with the cofactor-binding domain of its mesophilic counterpart CbADH [chimera Chi21((TCT))]. This domain exchange significantly destabilized the parent thermophilic enzyme (DeltaT(1/2) = -18 degrees C). The reverse exchange in CbADH [chimera Chi22((CTC))], however, had little effect on the thermal stability of the parent mesophilic protein. Furthermore, substituting the cofactor-binding domain of TbADH with the homologous domain of EhADH1 [chimera Chi23((TET))] substantially reduced the thermal stability of the thermophilic ADH (DeltaT(1/2) = -51 degrees C) and impeded the oligomerization of the enzyme. All three chimeric proteins and one of their site-directed mutants were crystallized, and their three-dimensional (3D) structures were determined. Comparison of the 3D structures of the chimeras and the chimeric mutant with the structures of their parent ADHs showed no significant changes to their Calpha chains, suggesting that the difference in the thermal stability of the three parent ADHs and their chimeric mutants could be due to a limited number of substitutions located at strategic positions, mainly at the oligomerization interfaces. Indeed, stabilization of the chimeras was achieved, to a significant extent, either by introduction of a proline residue at a strategic position in the major horse liver ADH-type dimerization interface (DeltaT(1/2) = 35 degrees C) or by introduction of intersubunit electrostatic interactions (DeltaT(1/2) = 6 degrees C). << Less

  Biochemistry 49:1943-1953(2010) [PubMed] [EuropePMC]