Purification and characterization of membrane-bound aldehyde dehydrogenase from Acetobacter polyoxogenes sp. nov.

Tayama, Kenji; Fukaya, Masahiro; Okumura, Hajime; Kawamura, Yoshiya; Beppu, Teruhiko

doi:10.1007/bf00165884

Cited by 39 publications

(23 citation statements)

References 9 publications

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“…The primary structure of AcDH-II was also compared with that of an AIDH from Acetobacter polyoxogenes (47); however, no relevant homologies were obtained. In contrast to the enzyme of A. eutrophus, the AIDH of the acetic acid bacterium is membrane bound and pyridine nucleotide independent (14).…”

Section: Methodsmentioning

confidence: 90%

Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus

et al. 1992

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The N-terminal amino acid sequence of purified acetaldehyde dehydrogenase II (AcDH-II) from ethanolgrown cells ofAlcaligenes eutrophus was determined. By using oligonucleotides deduced from this sequence the structural gene for AcDH-II, which was referred to as acoD, was localized on a 7.2-kbp EcoRI restriction fragment (fragment D), which has been cloned recently (C. Frund, H. Priefert, A. Steinbuchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989 The aerobic bacterium Alcaligenes eutrophus H16 synthesizes two different NAD-dependent acetaldehyde dehydrogenases (AcDH-I and AcDH-II; EC 1.2.1.3) during growth on acetoin. Both dehydrogenases are also synthesized in the type strain TF93 and in ethanol-utilizing mutants of A. eutrophus derived from strains H16 or N9A during growth on ethanol or on acetoin. Since the expression of AcDH-II is specifically induced only during growth on these substrates, this enzyme seems to be responsible for the oxidation of acetaldehyde to acetate in both catabolic pathways. AcDH-II possesses a high affinity toward acetaldehyde (Km = 4 ,uM), exhibits a wide substrate spectrum, and amounts to about 14% of the total soluble protein in ethanol-grown cells (21).In A. eutrophus the degradation of acetoin is initiated by direct cleavage into two C2 compounds (13). This reaction is catalyzed by acetoin:2,6-dichlorophenolindophenol oxidoreductase and occurs also in Bacillus subtilis (28) and in the anaerobic bacterium Pelobacter carbinolicus (33) during growth on acetoin. Recently, we have identified and localized the genes, which are essentially involved in the catabolism of acetoin in A. eutrophus, on five different genomic EcoRI restriction fragments (A, B, C, D, and E) (13). The structural genes for the a-and P-subunits of acetoin:2,6-dichlorophenolindophenol oxidoreductase (acoA and acoB, respectively); for the protein FMP (acoC), which seems to be a dihydrolipoamide acetyltransferase; and for a protein of yet unknown function (acoX) are probably organized in one single operon (acoXABC) which is localized on fragments A and C (36). Fragment B harbors an rpoN-like gene, and mutants carrying a TnS insertion in this gene exhibited an extremely pleiotropic phenotype (13,38). As these mutants * Corresponding author.were also unable to utilize acetoin as a sole carbon source for growth, the expression of acetoin-catabolic genes in A. eutrophus seems to be also rpoN dependent. This is consistent with the identification of a promoter exhibiting striking homology to the enterobacterial c54-dependent promoter consensus sequence upstream of acoX-ABC. The information encoded by fragments D and E remained to be elucidated.In the present study we localized the structural gene for AcDH-II (acoD), which is in A. eutrophus essentially involved in the catabolism of acetoin as well as of ethanol, on EcoRI restriction fragment D. The results of the molecular characterization of acoD and of adjacent DNA regions confirmed our assumption that the expression of acoD is also rpoN dependent, like the ex...

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Section: Methodsmentioning

confidence: 90%

Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus

et al. 1992

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“…However, three CXXCH sequence motifs for heme C binding site were identified in the deduced amino acid sequence of the 46-kDa subunit, and none were identified in the sequence of the 79-kDa subunit (29). The ALDH of A. polyoxogenes (9) was apparently purified as a defective complex containing one 75-kDa subunit and one 19-kDa subunit and was therefore lacking the mediumsize cytochrome c subunit (15). In addition, the one-subunit enzyme (125 kDa) purified from A. aceti was claimed to lack the typical absorption of a cytochrome c component (23).…”

Section: Discussionmentioning

confidence: 99%

Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus , a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b , and Cytochrome c

Gómez-Manzo¹,

Chávez‐Pacheco²,

Contreras-Zentella³

et al. 2010

J Bacteriol

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Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone.

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“…Therefore, the 15-kDa protein in the ADH complex in A. pasteurianus appears to play no crucial role in the ethanol-oxidizing activity. However, it would be interesting to clarify the function of the small subunit because several membrane-bound dehydrogenases of acetic acid bacteria also contain a similar small subunit (4,5,9,24). These observations suggest the involvement of this type of a small subunit in the electron transport system linked with the membrane.…”

Section: Discussionmentioning

confidence: 99%

Induction by ethanol of alcohol dehydrogenase activity in Acetobacter pasteurianus

et al. 1993

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The membrane-bound alcohol dehydrogenase (ADH) activity of Acetobacter pasteurianus NC11380 was enhanced more than 10-fold by the addition of ethanol to the medium. In order to elucidate the mechanism of the ethanol induction, a gene cluster encoding the dehydrogenase and cytochrome c subunits of ADH was cloned from this strain, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequences and the NH2-terminal sequences determined with purified proteins showed that the dehydrogenase and cytochrome c subunits contained typical signal peptides of 35 and 26 amino acids, respectively. Transcriptional analysis of the cloned genes by primer extension revealed that the gene cluster was transcribed from two different promoters upstream from the dehydrogenase gene. One (59 bp upstream of the ATG start codon) of the two promoters was used in the presence of ethanol, whereas the other (232 bp upstream of the ATG start codon) was used in the absence of ethanol. Immunoblot analyses showed that almost the same amounts of the cytochrome c and the 15-kDa subunits were produced in both the presence and absence of ethanol and that the amount of the dehydrogenase subunit localized in the membrane was decreased in the absence of ethanol. This incorrect localization of the dehydrogenase subunit might be one of the factors responsible for the low ADH activity in the absence of ethanol.The most characteristic process in vinegar production is acetic acid fermentation, in which ethanol is oxidized to acetic acid by acetic acid bacteria, now classified as Acetobacter and Gluconobacter spp. (8). This ethanol oxidation is catalyzed by two membrane-bound enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (1-4, 9, 27). ADHs of acetic acid bacteria have been purified as complexes composed of more than two subunits, including the dehydrogenase subunit and the cytochrome c subunit (1,3,27). Our recent genetic study of the ADH gene cluster from Acetobacter polyoxogenes NC11028 revealed that not only the dehydrogenase subunit but also the cytochrome c subunit of ADH were essential for ADH activity (26). Matsushita et al. (18) also drew a similar conclusion from reconstitution analyses for the cytochrome c subunit purified from Gluconobacter suboxydans into the membrane prepared from an ADH-defective strain of G. suboxydans.Cloning and sequencing analyses of ADH genes from A. polyoxogenes (26) and Acetobacter aceti (14,15) showed that in both species, genes encoding the dehydrogenase subunit and the cytochrome c subunit were clustered in the same transcription polarity. This structural feature of the ADH gene cluster suggested that the genes for both subunits formed an operon and were cotranscribed. On the other hand, Matsushita et al. (17) showed that the cytochrome c subunit of ADH from G. suboxydans had an additional function as a component of the non-energy-generating bypass of the sugar-oxidizing respiratory chain and that the amount of the cytochrome c subunit in the membrane markedly increased when ...

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Purification and characterization of membrane-bound aldehyde dehydrogenase from Acetobacter polyoxogenes sp. nov.

Cited by 39 publications

References 9 publications

Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus

Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus

Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus , a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b , and Cytochrome c

Induction by ethanol of alcohol dehydrogenase activity in Acetobacter pasteurianus

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