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J Bacteriol. 1994 January; 176(2): 469–485.
PMCID: PMC205071

Identification and molecular characterization of the aco genes encoding the Pelobacter carbinolicus acetoin dehydrogenase enzyme system.


Use of oligonucleotide probes, which were deduced from the N-terminal sequences of the purified enzyme components, identified the structural genes for the alpha and beta subunits of E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehydrogenase) of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system, which were designated acoA, acoB, acoC, and acoL, respectively. The nucleotide sequences of acoA (979 bp), acoB (1,014 bp), acoC (1,353 bp), and acoL (1,413 bp) as well as of acoS (933 bp), which encodes a protein with an M(r) of 34,421 exhibiting 64.7% amino acid identity to the Escherichia coli lipA gene product, were determined. These genes are clustered on a 6.1-kbp region. Heterologous expression of acoA, acoB, acoC, acoL, and acoS in E. coli was demonstrated. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 34,854), E1 beta (M(r), 36,184), E2 (M(r), 47,281), and E3 (M(r), 49,394) exhibited striking similarities to the amino acid sequences of the components of the Alcaligenes eutrophus acetoin-cleaving system. Homologies of up to 48.7% amino acid identity to the primary structures of the enzyme components of various 2-oxo acid dehydrogenase complexes also were found. In addition, the respective genes of the 2-oxo acid dehydrogenase complexes and of the acetoin dehydrogenase enzyme system were organized very similarly, indicating a close relationship of the P. carbinolicus acetoin dehydrogenase enzyme system to 2-oxo acid dehydrogenase complexes.

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  • Ausubel FM. Regulation of nitrogen fixation genes. Cell. 1984 May;37(1):5–6. [PubMed]
  • Behal RH, Browning KS, Reed LJ. Nucleotide and deduced amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase. Biochem Biophys Res Commun. 1989 Oct 31;164(2):941–946. [PubMed]
  • Benson AM, Mower HF, Yasunobu KT. The amino acid sequence of Clostridium butyricum ferredoxin. Proc Natl Acad Sci U S A. 1966 Jun;55(6):1532–1535. [PubMed]
  • Bibb MJ, Findlay PR, Johnson MW. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene. 1984 Oct;30(1-3):157–166. [PubMed]
  • Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. [PMC free article] [PubMed]
  • Bleile DM, Munk P, Oliver RM, Reed LJ. Subunit structure of dihydrolipoyl transacetylase component of pyruvate dehydrogenase complex from Escherichia coli. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4385–4389. [PubMed]
  • Borges A, Hawkins CF, Packman LC, Perham RN. Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Eur J Biochem. 1990 Nov 26;194(1):95–102. [PubMed]
  • Broome-Smith JK, Ioannidis I, Edelman A, Spratt BG. Nucleotide sequences of the penicillin-binding protein 5 and 6 genes of Escherichia coli. Nucleic Acids Res. 1988 Feb 25;16(4):1617–1617. [PMC free article] [PubMed]
  • Browner MF, Taroni F, Sztul E, Rosenberg LE. Sequence analysis, biogenesis, and mitochondrial import of the alpha-subunit of rat liver propionyl-CoA carboxylase. J Biol Chem. 1989 Jul 25;264(21):12680–12685. [PubMed]
  • Carlsson P, Hederstedt L. Genetic characterization of Bacillus subtilis odhA and odhB, encoding 2-oxoglutarate dehydrogenase and dihydrolipoamide transsuccinylase, respectively. J Bacteriol. 1989 Jul;171(7):3667–3672. [PMC free article] [PubMed]
  • Carothers DJ, Pons G, Patel MS. Dihydrolipoamide dehydrogenase: functional similarities and divergent evolution of the pyridine nucleotide-disulfide oxidoreductases. Arch Biochem Biophys. 1989 Feb 1;268(2):409–425. [PubMed]
  • Chang YY, Cronan JE, Jr, Li SJ, Reed K, Vanden Boom T, Wang AY. Locations of the lip, poxB, and ilvBN genes on the physical map of Escherichia coli. J Bacteriol. 1991 Sep;173(17):5258–5259. [PMC free article] [PubMed]
  • Davie JR, Wynn RM, Cox RP, Chuang DT. Expression and assembly of a functional E1 component (alpha 2 beta 2) of mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli. J Biol Chem. 1992 Aug 15;267(23):16601–16606. [PubMed]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [PMC free article] [PubMed]
  • Dietrichs D, Andreesen JR. Purification and comparative studies of dihydrolipoamide dehydrogenases from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and Clostridium sporogenes. J Bacteriol. 1990 Jan;172(1):243–251. [PMC free article] [PubMed]
  • DOLIN MI. Diacetyl oxidation by Streptococcus faecalis, a lipoic acid dependent reaction. J Bacteriol. 1955 Jan;69(1):51–58. [PMC free article] [PubMed]
  • Fitzgerald J, Hutchison WM, Dahl HH. Isolation and characterisation of the mouse pyruvate dehydrogenase E1 alpha genes. Biochim Biophys Acta. 1992 May 7;1131(1):83–90. [PubMed]
  • Fründ C, Priefert H, Steinbüchel A, Schlegel HG. Biochemical and genetic analyses of acetoin catabolism in Alcaligenes eutrophus. J Bacteriol. 1989 Dec;171(12):6539–6548. [PMC free article] [PubMed]
  • Grunstein M, Hogness DS. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. [PubMed]
  • Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. [PubMed]
  • Hanemaaijer R, de Kok A, Jolles J, Veeger C. The domain structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Eur J Biochem. 1987 Dec 1;169(2):245–252. [PubMed]
  • Hanemaaijer R, Janssen A, de Kok A, Veeger C. The dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Molecular cloning and sequence analysis. Eur J Biochem. 1988 Jul 1;174(4):593–599. [PubMed]
  • Hanna Z, Fregeau C, Préfontaine G, Brousseau R. Construction of a family of universal expression plasmid vectors. Gene. 1984 Oct;30(1-3):247–250. [PubMed]
  • Hawkins CF, Borges A, Perham RN. A common structural motif in thiamin pyrophosphate-binding enzymes. FEBS Lett. 1989 Sep 11;255(1):77–82. [PubMed]
  • Hawkins CF, Borges A, Perham RN. Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Eur J Biochem. 1990 Jul 31;191(2):337–346. [PubMed]
  • Hawley DK, McClure WR. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. [PMC free article] [PubMed]
  • Hayden MA, Huang I, Bussiere DE, Ashley GW. The biosynthesis of lipoic acid. Cloning of lip, a lipoate biosynthetic locus of Escherichia coli. J Biol Chem. 1992 May 15;267(14):9512–9515. [PubMed]
  • Hayden MA, Huang IY, Iliopoulos G, Orozco M, Ashley GW. Biosynthesis of lipoic acid: characterization of the lipoic acid auxotrophs Escherichia coli W1485-lip2 and JRG33-lip9. Biochemistry. 1993 Apr 13;32(14):3778–3782. [PubMed]
  • Hemilä H, Palva A, Paulin L, Arvidson S, Palva I. Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase. J Bacteriol. 1990 Sep;172(9):5052–5063. [PMC free article] [PubMed]
  • Herbert AA, Guest JR. Biochemical and genetic studies with lysine+methionine mutants of Escherichia coli: lipoic acid and alpha-ketoglutarate dehydrogenase-less mutants. J Gen Microbiol. 1968 Oct;53(3):363–381. [PubMed]
  • Hjelm H, Hjelm K, Sjöquist J. Protein A from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins. FEBS Lett. 1972 Nov 15;28(1):73–76. [PubMed]
  • Hohn B. In vitro packaging of lambda and cosmid DNA. Methods Enzymol. 1979;68:299–309. [PubMed]
  • Hohn B, Murray K. Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3259–3263. [PubMed]
  • Hu CW, Lau KS, Griffin TA, Chuang JL, Fisher CW, Cox RP, Chuang DT. Isolation and sequencing of a cDNA encoding the decarboxylase (E1)alpha precursor of bovine branched-chain alpha-keto acid dehydrogenase complex. Expression of E1 alpha mRNA and subunit in maple-syrup-urine-disease and 3T3-L1 cells. J Biol Chem. 1988 Jun 25;263(18):9007–9014. [PubMed]
  • Huh TL, Casazza JP, Huh JW, Chi YT, Song BJ. Characterization of two cDNA clones for pyruvate dehydrogenase E1 beta subunit and its regulation in tricarboxylic acid cycle-deficient fibroblast. J Biol Chem. 1990 Aug 5;265(22):13320–13326. [PubMed]
  • JUNI E, HEYM GA. A cyclic pathway for the bacterial dissimilation of 2, 3-butanediol, acetylmethylcarbinol, and diacetyl. I. General aspects of the 2, 3-butanediol cycle. J Bacteriol. 1956 Apr;71(4):425–432. [PMC free article] [PubMed]
  • Knauf VC, Nester EW. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. [PubMed]
  • Krüger N, Steinbüchel A. Identification of acoR, a regulatory gene for the expression of genes essential for acetoin catabolism in Alcaligenes eutrophus H16. J Bacteriol. 1992 Jul;174(13):4391–4400. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Leary JJ, Brigati DJ, Ward DC. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4045–4049. [PubMed]
  • López J, Thoms B, Fortnagel P. Mutants of Bacillus subtilis blocked in acetoin reductase. Eur J Biochem. 1973 Dec 17;40(2):479–483. [PubMed]
  • López JM, Thoms B, Rehbein H. Acetoin degradation in Bacillus subtilis by direct oxidative cleavage. Eur J Biochem. 1975 Sep 15;57(2):425–430. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Niu XD, Browning KS, Behal RH, Reed LJ. Cloning and nucleotide sequence of the gene for dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7546–7550. [PubMed]
  • Nobukuni Y, Mitsubuchi H, Endo F, Akaboshi I, Asaka J, Matsuda I. Maple syrup urine disease. Complete primary structure of the E1 beta subunit of human branched chain alpha-ketoacid dehydrogenase complex deduced from the nucleotide sequence and a gene analysis of patients with this disease. J Clin Invest. 1990 Jul;86(1):242–247. [PMC free article] [PubMed]
  • Oppermann FB, Schmidt B, Steinbüchel A. Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system. J Bacteriol. 1991 Jan;173(2):757–767. [PMC free article] [PubMed]
  • Oppermann FB, Steinbüchel A, Schlegel HG. Evidence for oxidative thiolytic cleavage of acetoin in Pelobacter carbinolicus analogous to aerobic oxidative decarboxylation of pyruvate. FEMS Microbiol Lett. 1989 Jul 1;51(1):113–118. [PubMed]
  • Otulakowski G, Robinson BH. Isolation and sequence determination of cDNA clones for porcine and human lipoamide dehydrogenase. Homology to other disulfide oxidoreductases. J Biol Chem. 1987 Dec 25;262(36):17313–17318. [PubMed]
  • Palmer JA, Hatter K, Sokatch JR. Cloning and sequence analysis of the LPD-glc structural gene of Pseudomonas putida. J Bacteriol. 1991 May;173(10):3109–3116. [PMC free article] [PubMed]
  • Palmer JA, Madhusudhan KT, Hatter K, Sokatch JR. Cloning, sequence and transcriptional analysis of the structural gene for LPD-3, the third lipoamide dehydrogenase of Pseudomonas putida. Eur J Biochem. 1991 Dec 5;202(2):231–240. [PubMed]
  • Perham RN, Packman LC. 2-Oxo acid dehydrogenase multienzyme complexes: domains, dynamics, and design. Ann N Y Acad Sci. 1989;573:1–20. [PubMed]
  • Perham RN, Packman LC, Radford SE. 2-Oxo acid dehydrogenase multi-enzyme complexes: in the beginning and halfway there. Biochem Soc Symp. 1987;54:67–81. [PubMed]
  • Priefert H, Hein S, Krüger N, Zeh K, Schmidt B, Steinbüchel A. Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism. J Bacteriol. 1991 Jul;173(13):4056–4071. [PMC free article] [PubMed]
  • Reed KE, Cronan JE., Jr Lipoic acid metabolism in Escherichia coli: sequencing and functional characterization of the lipA and lipB genes. J Bacteriol. 1993 Mar;175(5):1325–1336. [PMC free article] [PubMed]
  • Reed LJ, Hackert ML. Structure-function relationships in dihydrolipoamide acyltransferases. J Biol Chem. 1990 Jun 5;265(16):8971–8974. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Stephens PE, Darlison MG, Lewis HM, Guest JR. The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component. Eur J Biochem. 1983 Jun 1;133(1):155–162. [PubMed]
  • Stephens PE, Darlison MG, Lewis HM, Guest JR. The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the dihydrolipoamide acetyltransferase component. Eur J Biochem. 1983 Jul 1;133(3):481–489. [PubMed]
  • Strauss EC, Kobori JA, Siu G, Hood LE. Specific-primer-directed DNA sequencing. Anal Biochem. 1986 Apr;154(1):353–360. [PubMed]
  • Studier FW. Genetic mapping of a mutation that causes ribonucleases III deficiency in Escherichia coli. J Bacteriol. 1975 Oct;124(1):307–316. [PMC free article] [PubMed]
  • Tanaka M, Nakashima T, Benson A, Mower H, Tasunobu KT. The amino acid sequence of Clostridium pasteurianum ferredoxin. Biochemistry. 1966 May;5(5):1666–1681. [PubMed]
  • Tinoco I, Jr, Borer PN, Dengler B, Levin MD, Uhlenbeck OC, Crothers DM, Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PubMed]
  • Vanden Boom TJ, Reed KE, Cronan JE., Jr Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, and identification of the lipoylated protein of the glycine cleavage system. J Bacteriol. 1991 Oct;173(20):6411–6420. [PMC free article] [PubMed]
  • Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. [PubMed]
  • Vogelstein B, Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. [PubMed]
  • Westphal AH, de Kok A. Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene. Eur J Biochem. 1988 Mar 1;172(2):299–305. [PubMed]
  • Williams OB, Morrow MB. THE BACTERIAL DESTRUCTION OF ACETYL-METHYL-CARBINOL. J Bacteriol. 1928 Jul;16(1):43–48. [PMC free article] [PubMed]
  • Wynn RM, Chuang JL, Davie JR, Fisher CW, Hale MA, Cox RP, Chuang DT. Cloning and expression in Escherichia coli of mature E1 beta subunit of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex. Mapping of the E1 beta-binding region on E2. J Biol Chem. 1992 Jan 25;267(3):1881–1887. [PubMed]

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