Enzymatic investigations on butanol dehydrogenase and butyraldehyde dehydrogenase in extracts of Clostridium acetobutylicum

Dürre, Peter; Kuhn, Anita; Gottwald, Matthias; Gottschalk, Gerhard

doi:10.1007/bf00286322

Cited by 79 publications

(41 citation statements)

References 13 publications

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“…The solvent-producing clostridia also contain multiple ADHs. Clostndium acetobutylicum has at least three different primary ADHs (4,14,44,54), and the amino acid sequences encoded by adhl (61) and bdhA and bdhB (52) have been deduced. C. beijerinckii has a primary-secondary ADH (reference 19 and this study) and multiple primary ADHs with different coenzyme specificities (24,57).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 1993

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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...

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Section: Discussionmentioning

confidence: 99%

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

et al. 1993

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“…The ADH enzyme assays were based on described methods (30,31). Briefly, half-liter cultures of each strain were grown to OD 600 = 0.6 and centrifuged at 4°C at 6,000 × g in a Beckman Coulter Avanti J-25 centrifuge with a JA-10 rotor.…”

Section: Analysis Of Distribution Of Mutations Inferred From Pyrosequmentioning

confidence: 99%

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Brown

Guss

Karpinets

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

167

145

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Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene ( adhE ), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

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“…In this step, butyraldehyde is converted to butanol, with the cofactor NAD(P)H being oxidized in the process. Evidence suggests that there are two distinct types of BDHs in C. acetobutylicum, one which is NADH dependent and one that is NADPH dependent (11,25). The relative physiological importance of these two dehydrogenases is unclear at present.…”

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confidence: 99%

Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes

1992

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A 4-kb segment of DNA containing two previously cloned butanol dehydrogenase (BDH) isozyme genes (D.Petersen, R. Welch, F. Rudolph, and G. Bennett, J. Bacteriol. 173:1831-1834 was sequenced. Two complete open reading frames (ORFs) were identified (bdhA and bdhB), along with a third truncated ORF (ORF1). The translation products of bdka4 and bdhB corresponded to the N-terminal sequences of the purified BDH I and BDH H proteins, respectively. The two isozymes had a high amino acid identity (73%) and showed homology to a newly described class of alcohol dehydrogenases. Northern blots revealed that bdhA and bdhB did not form an operon. Primer extension experiments located single transcriptional start sites 37 and 58 bp upstream of the start codons of bdhA and bdhB, respectively. The -10 and -35 promoter regions for these genes were almost identical. bMUA and bdhB were found to be induced or derepressed immediately prior to significant butanol production in controlled pH 5.0 batch fermentations.The gram-positive, obligately anaerobic bacterium Clostridium acetobutylicum is one of the few organisms known to produce 1-butanol as a major fermentation product. Interest in the use of butanol as a fuel extender and chemical feedstock has prompted several investigations into the molecular means by which C acetobutylicum produces butanol.The butanol dehydrogenase (BDH) is involved in the final step of the butanol formation pathway in C. acetobutylicum. In this step, butyraldehyde is converted to butanol, with the cofactor NAD(P)H being oxidized in the process. Evidence suggests that there are two distinct types of BDHs in C. acetobutylicum, one which is NADH dependent and one that is NADPH dependent (11,25). The relative physiological importance of these two dehydrogenases is unclear at present. Previously, Youngleson et al. (42,43) cloned and sequenced the adhl gene from C. acetobutylicum P262. This gene codes for a 43-kDa NADPH-dependent alcohol dehydrogenase (ADH). No activity was observed with NADH as a cofactor. The enzyme utilized ethanol and butanol nearly equally well and was thus classified as an ADH.More recently, two BDH isozymes (BDH I, BDH II) were purified and cloned from C. acetobutylicum ATCC 824 (25,35,36). These isozymes were primarily NADH dependent, although some NADPH-dependent activity was observed. Both enzymes were dimers with a subunit molecular size of -42 kDa. The BDH II enzyme was found to have 46-fold greater activity with butyraldehyde than with acetaldehyde, whereas the BDH I enzyme had only 2-fold greater activity.In this study, the nucleotide sequence and putative amino acid sequence of the bdhA and bdhB genes are presented.These two genes code for proteins showing homology to a newly described class of ADHs. Although the bdhA and bdhB genes were found to exist in tandem on the chromosome, RNA studies revealed that they do not form an operon. In addition, both genes were shown to be induced or * Corresponding author. derepressed near the onset of butanol formation in batch culture. MATERIALS ...

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Enzymatic investigations on butanol dehydrogenase and butyraldehyde dehydrogenase in extracts of Clostridium acetobutylicum

Cited by 79 publications

References 13 publications

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

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

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes

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