Utilization of methylacetoin by the strict anaerobe Pelobacter carbinolicus and consequences for the catabolism of acetoin

Oppermann, Fred Bernd

doi:10.1016/0378-1097(88)90149-8

Cited by 12 publications

(32 citation statements)

References 5 publications

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“…dominant protein band by activity staining after nondenaturing PAGE, whereas this protein was absent in the protein pattern of ethylene glycol-grown cells (36). Analogous results were obtained with crude extracts of P. venetianus and P. acetylenicus.…”

Section: Downloaded Fromsupporting

confidence: 69%

“…For the catabolism of acetoin in P. carbinolicus, four possible pathways were discussed. Physiological and enzymatic investigations in addition to energetic considerations provided evidence that in P. carbinolicus acetoin is most likely cleaved into acetyl-CoA and acetaldehyde by an oxidative-thiolytic overall reaction (36). In crude extracts from acetoin-grown cells of P. carbinolicus, a CoASH-and NAD-dependent oxidative cleavage of acetoin into acetaldehyde and acetyl-CoA and of methylacetoin into acetone and acetyl-CoA has been demonstrated recently (37).…”

Section: Discussionmentioning

confidence: 97%

“…Since no pyruvate-or 2-oxoglutarate-dependent reduction of DCPIP was measured with crude extracts and with purified Ao:DCPIP OR, it is unlikely that the cleavage of acetoin is catalyzed by nonspecific E1 components of 2-oxo acid dehydrogenase complexes. The in vitro cleavage of diacetyl into stoichiometric amounts of acetate, which is catalyzed by Ao:DCPIP OR, seems to have no physiological relevance, as revealed from considerations of energy and from physiological experiments (36). In gas chromatographic analysis of the products of acetoin conversion in crude extracts in the presence of NAD and coenzyme A and with purified Ao:DCPIP OR in the presence of DCPIP, diacetyl could not be detected.…”

Section: Discussionmentioning

confidence: 99%

“…DL-Dihydrolipoamide was prepared by reduction of DL-lipoamide as described before (42). Ao:DCPIP OR was assayed photometrically at 578 nm as described previously (36). Alcohol dehydrogenase (EC 1.1.1.1), 2,3-butanediol dehydrogenase (EC 1.1.1.4), malate dehydrogenase (EC 1.1.1.37), and acetate kinase (EC 2.7.2.1) were determined by measuring changes in A365 nm with NAD and NADH, respectively (4,57).…”

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confidence: 99%

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Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system

1991

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Dihydrolipoamide dehydrogenase (DHLDH), dihydrolipoamide acetyltransferase (DHLTA), and acetoin: 2,6-dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) were purified from acetoin-grown cells of Pelobacter carbinolicus. DHLDH had a native Mr of 110,000, consisted of two identical subunits of Mr 54,000, and reacted only with NAD(H) as a coenzyme. The N-terminal amino acid sequence included the flavin adenine dinucleotide-binding site and exhibited a high degree of homology to other DHLDHs. DHLTA had a native Mr of greater than 500,000 and consisted of subunits identical in size (Mr 60,000). The enzyme was highly sensitive to proteolytic attack. During limited tryptic digestion, two major fragments of Mr 32,500 and 25,500 were formed. Ao:DCPIP OR consisted of two different subunits of Mr 37,500 and 38,500 and had a native Mr in the range of 143,000 to 177,000. In vitro in the presence of DCPIP, it catalyzed a thiamine pyrophosphate-dependent oxidative-hydrolytic cleavage of acetoin, methylacetoin, and diacetyl. The combination of purified Ao:DCPIP OR, DHLTA, and DHLDH in the presence of thiamine pyrophosphate and the substrate acetoin or methylacetoin resulted in a coenzyme A-dependent reduction of NAD. In the strictly anaerobic acetoin-utilizing bacteria P. carbinolicus, Pelobacter venetianus, Pelobacter acetylenicus, Pelobacter propionicus, Acetobacterium carbinolicum, and Clostridium magnum, the enzymes Ao:DCPIP OR, DHLTA, and DHLDH were induced during growth on acetoin, whereas they were absent or scarcely present in cells grown on a nonacetoinogenic substrate.

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Section: Downloaded Fromsupporting

confidence: 69%

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system

1991

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“…Therefore, this bacterium lacks the 2,3-butanedi oil cycle which was described by Juni and Heym (36) in Aci netobacter calcoaceticus. In contrast, A. eutrophus degr&Ldes acetoin directly by cleavage into two C2 compounds , like Bacillus subtilis (35,43,44) or Pelobacter car binolicus (48)(49)(50).…”

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Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism

et al. 1991

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Acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) and the fast-migrating protein (FMP) were purified to homogeneity from crude ex tracts of acetoin-grown cells of Alcaligenes eutrophus. Ao:DCPIP OR consisted of a and , subunits (Mrs, 35,5 00 and 36,000, respectively), and a tetrameric a2,12 structure was most likely for the native protein. The mole cular weight of FMP subunits was 39,000. The N-terminal amino acid sequences of the three proteins were det ermined, and oligonucleotides were synthesized on the basis of the codon usage of A. eutrophus. With these, the structural genes for the a and ,1 subunits of Ao:DCPIP OR and FMP, which were referred to as acoA, aco)B, and acoC, respectively, were localized on one single EcoRI restriction fragment which has been clonei recently (C. Frund, H. Priefert, A. Steinbuchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The nucleotide sequences of a 5.3-kbp region of this fragment and one adjacent fragment were determined, an d the structural genes for acoA (1,002 bp), acoB (1,017 bp), and acoC (1,125 bp) were identified. Together woith the gene acoX, whose function is still unknown and which is represented by a 1,080-bp open reading fr ame, these genes are probably organized in one single operon (acoXABC). The transcription start site was identified 27 bp upstream of acoX; this site was preceded by a region which exhibited complete homology to the enterobacterial a54-dependent promoter consensus sequence.The amino acid sequences deduced from aco. 4 and acoB for the a subunit (Mr,35,243) and the 0 subunit (Mr, 35,788) exhibited significant homologies to th e primary structures of the dehydrogenase components of various 2-oxo acid dehydrogenase complexes, whei 'eas those deduced from acoC for FMP (Mr, 38,941) revealed homology to the dihydrolipoamide acetyltrarw,ferase of Escherichia coli. The occurrence of a new enzyme type for the degradation of acetoin is discussed.Alcaligenes eutrophus has been studied with respe ct to the degradation of acetoin. Acetoin-grown cells of A. eb rtrophus are devoid of 2,3-butanediol dehydrogenase and acetoin dehydrogenase (diacetyl forming; EC 1.1.1.5), and mutants which lack 2,3-butanediol dehydrogenase (73) are stilil able to utilize acetoin as the sole carbon source for grovvth (72). Therefore, this bacterium lacks the 2,3-butanedi oil cycle which was described by Juni and Heym (36) (23,59). As insertional inactivation of this gene caused a pleiotropic effect and as these mutants were unable to synthesize proteins which are essential for degradation of acetoin, it was concluded that expression of the corresponding genes is rpoN dependent (23).Physiological studies localized the genes for Ao:DCPIP OR and FMP on restriction fragments A and C, which are closely linked in the genome (23), whereas the gene for acetaldehyde dehydrogenase II was localized on restriction fragment D (52a). The present study was aimed at isolation of FMP and Ao:DCPIP OR and identification and characterization of their structural genes. ...

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Evidence for oxidative thiolytic cleavage of acetoin in Pelobacter carbinolicus analogous to aerobic oxidative decarboxylation of pyruvate

Oppermann¹

1989

FEMS Microbiology Letters

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Dihydrolipoamide dehydrogenase and dihydrolipoamide acetyltransferase were formed when Pelobacter carbinolicus strain GraBd1 was grown on acetoin. The specific activities of these enzymes amounted to 0.50 and 28.7 U/mg protein, respectively. The crude extract catalyzed the CoASH- and NAD+-dependent formation of acetyl-CoA from acetoin and methylacetoin. From ethylene glycol-grown cells these activities were absent. Crude extracts also exhibited acetoin: methyl viologen and acetoin: metronidazole oxidoreductase activity. As shown by reconstitution experiments methylviologen reduction was dependent on the presence of a light-brownish protein (Mr 220,000 +/- 10,000); metronidazole reduction was in addition dependent on the presence of a dark-brownish protein (Mr 4,900 +/- 800), which is probably a ferredoxin. However, both components were synthesized constitutively. We discussed a model for oxidative-thiolytic cleavage of acetoin which is analogous to the reaction of the pyruvate dehydrogenase enzyme complex rather than to pyruvate: ferredoxin oxidoreductase.

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Utilization of methylacetoin by the strict anaerobe Pelobacter carbinolicus and consequences for the catabolism of acetoin

Cited by 12 publications

References 5 publications

Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system

Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system

Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism

Evidence for oxidative thiolytic cleavage of acetoin in Pelobacter carbinolicus analogous to aerobic oxidative decarboxylation of pyruvate

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