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 backbone dynamics of the C-terminal DNA-binding domain of Escherichia coli topoisomerase I has been characterized in the absence and presence of single-stranded DNA by NMR spectroscopy. 15N spin-lattice relaxation times (T1), spin-spin relaxation times (T2), and heteronuclear NOEs were determined for the uniformly 15N-labeled protein. These data were analyzed by using the model-free formalism to derive the model-free parameters (S2, tau e, and R(ex)) for each backbone N-H bond vector and the overall molecular rotational correlation time (tau m)., The molecular rotational correlation time tau m was determined to be 7.49 +/- 0.36 ns for the free and 12.7 +/- 1.07 ns for the complexed protein. Several residues were found to be much more mobile than the average, including 11 residues at the N-terminus, 2 residues at the C-terminus, and residues 25 and 31-35 which are located in a region of the protein that binds to DNA. The binding of ssDNA to the free protein causes a slight increase in the order parameters (S2) for a small number of residues and a slight decrease in the order parameters (S2) for the majority of the residues. In particular, upon binding to ssDNA, the mobility of the first alpha-helix and the two beta-sheets was slightly increased, and the mobility of a few specific residues in the loops/turns was restricted. These results differ from the previous studies on the backbone dynamics of molecular complexes in which reduced mobilities were typically observed upon ligand binding.

The acidic residues Asp-111, Asp-113, and Glu-115 of Escherichia coli DNA topoisomerase I are located near the active site Tyr-319 and are conserved in type IA topoisomerase sequences with counterparts in type IIA DNA topoisomerases. Their exact functional roles in catalysis have not been clearly defined. Mutant enzymes with two or more of these residues converted to alanines were found to have >90% loss of activity in the relaxation assay with 6 mM Mg(II) present. Mg(II) concentrations (15-20 mM) inhibitory for the wild type enzyme are needed by these double mutants for maximal relaxation activity. The triple mutant D111A/D113A/E115A had no detectable relaxation activity. Mg(II) binding to wild type enzyme resulted in an altered conformation detectable by Glu-C proteolytic digestion. This conformational change was not observed for the triple mutant or for the double mutant D111A/D113A. Direct measurement of Mg(II) bound showed the loss of 1-2 Mg(II) ions for each enzyme molecule due to the mutations. These results demonstrate a functional role for these acidic residues in the binding of Mg(II) to induce the conformational change required for the relaxation of supercoiled DNA by the enzyme.Escherichia coli DNA topoisomerase I is the best studied representative of the type IA DNA topoisomerases. This class of enzymes includes the bacterial and archeal DNA topoisomerase I and III, reverse gyrase, and yeast and mammalian topoisomerase III, with diverse roles in cellular functions (reviewed in Refs. 1 and 2). Mg(II) is required for the interconversion of DNA topological isomers catalyzed by these enzymes. Comparison of their polypeptide sequences showed that the conserved positions include the acidic residues Asp-111, 4). When the crystal structure of the 67-kDa Nterminal transesterification domain of the enzyme was published, it was noted (5) that these three acidic residues in the active site are arranged similarly to the three acidic residues known to coordinate two divalent ions in Klenow fragment (6) that are required for the nucleotidyl transfer activity (7,8). These residues are found in domain I of the 67-kDa structure (5), which is similar to the BЈ domain of the Saccharomyces cerevisiae DNA topoisomerase II structure (9). There are corresponding acidic residues that are conserved in type IIA DNA topoisomerases (9). Severe loss of DNA relaxation and cleavage activities resulted when one of these acidic triad residues in S. cerevisiae DNA topoisomerase II, Asp-530, was mutated (10). Another conserved glutamate at Glu-9 of E. coli DNA topoisomerase I and the aspartates motif DXD at Asp-111 and Asp-113 have been proposed to be conserved motifs in a catalytic domain named Toprim found in type IA and IIA topoisomerases, as well as a number of other nucleotidyl transferases and polynucleotide cleaving activities (11). However, results of site-directed mutagenesis in E. coli DNA topoisomerase I showed that conversion of a single one of these three conserved acidic residues to alanine did not abolish the relaxation...

Escherichia coli DNA topoisomerase I catalyzes the interconversion of different topological forms of DNA. In this paper we describe NMR studies of a 14K C-terminal fragment of this enzyme that binds preferentially to single-stranded DNA and enhances the enzyme's ability to relax negatively supercoiled DNA under high salt conditions. The 1H, 13C, and 15N resonances of the protein were assigned from a number of heteronuclear multidimensional NMR experiments, and the three-dimensional structure of the protein was determined from a total of 2188 NMR-derived restraints. The root-mean-square deviation about the mean coordinate positions for residues 13-120 is 0.68 +/- 0.11 A for the backbone atoms and 1.09 +/- 0.09 A for all heavy atoms. The overall fold, which consists of two four-stranded beta-sheets separated by two helices, differs from other DNA- and RNA-binding proteins such as gene 5, cold shock protein, and hnRNP C. From an analysis of the changes in chemical shift upon the addition of single-stranded DNA, the location of the oligonucleotide binding site was determined. The binding site consists of a beta-sheet containing positively charged and aromatic amino acids and, in spite of its different structure, is similar to that found in other proteins that bind single-stranded oligonucleotides.