Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Jan;177(1):183–190. doi: 10.1128/jb.177.1.183-190.1995

Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli.

D D Skinner 1, M R Morgenstern 1, R W Fedechko 1, C D Denoya 1
PMCID: PMC176571  PMID: 8002616

Abstract

A cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH) of Streptomyces avermitilis has been cloned and sequenced. Open reading frame 1 (ORF1) (E1 alpha), 1,146 nucleotides long, would encode a polypeptide of 40,969 Da (381 amino acids). ORF2 (E1 beta), 1,005 nucleotides long, would encode a polypeptide of 35,577 Da (334 amino acids). The intergenic distance between ORF1 and ORF2 is 73 bp. The putative ATG start codon of the incomplete ORF3 (E2) overlaps the stop codon of ORF2. Computer-aided searches showed that the deduced products of ORF1 and ORF2 resembled the corresponding E1 subunit (alpha or beta) of several prokaryotic and eukaryotic BCDH complexes. When these ORFs were overexpressed in Escherichia coli, proteins of about 41 and 34 kDa, which are the approximate masses of the predicted S. avermitilis ORF1 and ORF2 products, respectively, were detected. In addition, specific E1 [alpha beta] BCDH activity was detected in E. coli cells carrying the S. avermitilis ORF1 (E1 alpha) and ORF2 (E1 beta) coexpressed under the control of the T7 promoter.

Full Text

The Full Text of this article is available as a PDF (327.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baylis H. A., Bibb M. J. The nucleotide sequence of a 16S rRNA gene from Streptomyces coelicolor A3(2) Nucleic Acids Res. 1987 Sep 11;15(17):7176–7176. doi: 10.1093/nar/15.17.7176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Behal R. H., Browning K. S., Reed L. J. 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. doi: 10.1016/0006-291x(89)91549-0. [DOI] [PubMed] [Google Scholar]
  3. Berg C. M., Vartak N. B., Wang G., Xu X., Liu L., MacNeil D. J., Gewain K. M., Wiater L. A., Berg D. E. The m gamma delta-1 element, a small gamma delta (Tn1000) derivative useful for plasmid mutagenesis, allele replacement and DNA sequencing. Gene. 1992 Apr 1;113(1):9–16. doi: 10.1016/0378-1119(92)90664-b. [DOI] [PubMed] [Google Scholar]
  4. Bibb M. J., Cohen S. N. Gene expression in Streptomyces: construction and application of promoter-probe plasmid vectors in Streptomyces lividans. Mol Gen Genet. 1982;187(2):265–277. doi: 10.1007/BF00331128. [DOI] [PubMed] [Google Scholar]
  5. Bibb M. J., Findlay P. R., Johnson M. W. 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. doi: 10.1016/0378-1119(84)90116-1. [DOI] [PubMed] [Google Scholar]
  6. Binnie C., Warren M., Butler M. J. Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis. J Bacteriol. 1989 Feb;171(2):887–895. doi: 10.1128/jb.171.2.887-895.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  9. Burg R. W., Miller B. M., Baker E. E., Birnbaum J., Currie S. A., Hartman R., Kong Y. L., Monaghan R. L., Olson G., Putter I. Avermectins, new family of potent anthelmintic agents: producing organism and fermentation. Antimicrob Agents Chemother. 1979 Mar;15(3):361–367. doi: 10.1128/aac.15.3.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Burns G., Brown T., Hatter K., Idriss J. M., Sokatch J. R. Similarity of the E1 subunits of branched-chain-oxoacid dehydrogenase from Pseudomonas putida to the corresponding subunits of mammalian branched-chain-oxoacid and pyruvate dehydrogenases. Eur J Biochem. 1988 Sep 15;176(2):311–317. doi: 10.1111/j.1432-1033.1988.tb14283.x. [DOI] [PubMed] [Google Scholar]
  11. Burns G., Brown T., Hatter K., Sokatch J. R. Comparison of the amino acid sequences of the transacylase components of branched chain oxoacid dehydrogenase of Pseudomonas putida, and the pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli. Eur J Biochem. 1988 Sep 1;176(1):165–169. doi: 10.1111/j.1432-1033.1988.tb14264.x. [DOI] [PubMed] [Google Scholar]
  12. Cook K. G., Bradford A. P., Yeaman S. J., Aitken A., Fearnley I. M., Walker J. E. Regulation of bovine kidney branched-chain 2-oxoacid dehydrogenase complex by reversible phosphorylation. Eur J Biochem. 1984 Dec 17;145(3):587–591. doi: 10.1111/j.1432-1033.1984.tb08597.x. [DOI] [PubMed] [Google Scholar]
  13. Dahl H. H., Brown R. M., Hutchison W. M., Maragos C., Brown G. K. A testis-specific form of the human pyruvate dehydrogenase E1 alpha subunit is coded for by an intronless gene on chromosome 4. Genomics. 1990 Oct;8(2):225–232. doi: 10.1016/0888-7543(90)90275-y. [DOI] [PubMed] [Google Scholar]
  14. Davie J. R., Wynn R. M., Cox R. P., Chuang D. T. 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] [Google Scholar]
  15. Denoya C. D., Bechhofer D. H., Dubnau D. Translational autoregulation of ermC 23S rRNA methyltransferase expression in Bacillus subtilis. J Bacteriol. 1986 Dec;168(3):1133–1141. doi: 10.1128/jb.168.3.1133-1141.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Denoya C. D., Skinner D. D., Morgenstern M. R. A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli. J Bacteriol. 1994 Sep;176(17):5312–5319. doi: 10.1128/jb.176.17.5312-5319.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dybvig K., Hollingshead S. K., Heath D. G., Clewell D. B., Sun F., Woodard A. Degenerate oligonucleotide primers for enzymatic amplification of recA sequences from gram-positive bacteria and mycoplasmas. J Bacteriol. 1992 Apr;174(8):2729–2732. doi: 10.1128/jb.174.8.2729-2732.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fisher C. W., Chuang J. L., Griffin T. A., Lau K. S., Cox R. P., Chuang D. T. Molecular phenotypes in cultured maple syrup urine disease cells. Complete E1 alpha cDNA sequence and mRNA and subunit contents of the human branched chain alpha-keto acid dehydrogenase complex. J Biol Chem. 1989 Feb 25;264(6):3448–3453. [PubMed] [Google Scholar]
  20. Fitzgerald J., Hutchison W. M., Dahl H. H. Isolation and characterisation of the mouse pyruvate dehydrogenase E1 alpha genes. Biochim Biophys Acta. 1992 May 7;1131(1):83–90. doi: 10.1016/0167-4781(92)90102-6. [DOI] [PubMed] [Google Scholar]
  21. Gould S. J., Subramani S., Scheffler I. E. Use of the DNA polymerase chain reaction for homology probing: isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1934–1938. doi: 10.1073/pnas.86.6.1934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hafner E. W., Holley B. W., Holdom K. S., Lee S. E., Wax R. G., Beck D., McArthur H. A., Wernau W. C. Branched-chain fatty acid requirement for avermectin production by a mutant of Streptomyces avermitilis lacking branched-chain 2-oxo acid dehydrogenase activity. J Antibiot (Tokyo) 1991 Mar;44(3):349–356. doi: 10.7164/antibiotics.44.349. [DOI] [PubMed] [Google Scholar]
  23. Hawkins C. F., Borges A., Perham R. N. A common structural motif in thiamin pyrophosphate-binding enzymes. FEBS Lett. 1989 Sep 11;255(1):77–82. doi: 10.1016/0014-5793(89)81064-6. [DOI] [PubMed] [Google Scholar]
  24. Hawkins C. F., Borges A., Perham R. N. 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. doi: 10.1111/j.1432-1033.1990.tb19128.x. [DOI] [PubMed] [Google Scholar]
  25. 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. doi: 10.1128/jb.172.9.5052-5063.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  27. Ho L., Patel M. S. Cloning and cDNA sequence of the beta-subunit component of human pyruvate dehydrogenase complex. Gene. 1990 Feb 14;86(2):297–302. doi: 10.1016/0378-1119(90)90294-2. [DOI] [PubMed] [Google Scholar]
  28. Hu C. W., Lau K. S., Griffin T. A., Chuang J. L., Fisher C. W., Cox R. P., Chuang D. T. 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] [Google Scholar]
  29. Johnson K. R., Komuniecki R., Sun Y., Wheelock M. J. Characterization of cDNA clones for the alpha subunit of pyruvate dehydrogenase from Ascaris suum. Mol Biochem Parasitol. 1992 Mar;51(1):37–47. doi: 10.1016/0166-6851(92)90198-s. [DOI] [PubMed] [Google Scholar]
  30. Koike K., Urata Y., Matsuo S., Koike M. Characterization and nucleotide sequence of the gene encoding the human pyruvate dehydrogenase alpha-subunit. Gene. 1990 Sep 14;93(2):307–311. doi: 10.1016/0378-1119(90)90241-i. [DOI] [PubMed] [Google Scholar]
  31. Kornberg H. L. The role and control of the glyoxylate cycle in Escherichia coli. Biochem J. 1966 Apr;99(1):1–11. doi: 10.1042/bj0990001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lowe P. N., Hodgson J. A., Perham R. N. Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis. Biochem J. 1983 Oct 1;215(1):133–140. doi: 10.1042/bj2150133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McCully V., Burns G., Sokatch J. R. Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO. Biochem J. 1986 Feb 1;233(3):737–742. doi: 10.1042/bj2330737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McKean M. C., Winkeler K. A., Danner D. J. Nucleotide sequence of the 5' end including the initiation codon of cDNA for the E1 alpha subunit of the human branched chain alpha-ketoacid dehydrogenase complex. Biochim Biophys Acta. 1992 Nov 15;1171(1):109–112. doi: 10.1016/0167-4781(92)90149-t. [DOI] [PubMed] [Google Scholar]
  35. Miran S. G., Lawson J. E., Reed L. J. Characterization of PDH beta 1, the structural gene for the pyruvate dehydrogenase beta subunit from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1252–1256. doi: 10.1073/pnas.90.4.1252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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. doi: 10.1172/JCI114690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Odessey R. Purification of rat kidney branched-chain oxo acid dehydrogenase complex with endogenous kinase activity. Biochem J. 1982 Apr 15;204(1):353–356. doi: 10.1042/bj2040353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Oku H., Kaneda T. Biosynthesis of branched-chain fatty acids in Bacillus subtilis. A decarboxylase is essential for branched-chain fatty acid synthetase. J Biol Chem. 1988 Dec 5;263(34):18386–18396. [PubMed] [Google Scholar]
  39. Paxton R., Harris R. A. Isolation of rabbit liver branched chain alpha-ketoacid dehydrogenase and regulation by phosphorylation. J Biol Chem. 1982 Dec 10;257(23):14433–14439. [PubMed] [Google Scholar]
  40. Paxton R., Kuntz M., Harris R. A. Phosphorylation sites and inactivation of branched-chain alpha-ketoacid dehydrogenase isolated from rat heart, bovine kidney, and rabbit liver, kidney, heart, brain, and skeletal muscle. Arch Biochem Biophys. 1986 Jan;244(1):187–201. doi: 10.1016/0003-9861(86)90108-6. [DOI] [PubMed] [Google Scholar]
  41. Perham R. N. Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes: a paradigm in the design of a multifunctional protein. Biochemistry. 1991 Sep 3;30(35):8501–8512. doi: 10.1021/bi00099a001. [DOI] [PubMed] [Google Scholar]
  42. Pettit F. H., Yeaman S. J., Reed L. J. Purification and characterization of branched chain alpha-keto acid dehydrogenase complex of bovine kidney. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4881–4885. doi: 10.1073/pnas.75.10.4881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Richardson M. A., Kuhstoss S., Solenberg P., Schaus N. A., Rao R. N. A new shuttle cosmid vector, pKC505, for streptomycetes: its use in the cloning of three different spiramycin-resistance genes from a Streptomyces ambofaciens library. Gene. 1987;61(3):231–241. doi: 10.1016/0378-1119(87)90187-9. [DOI] [PubMed] [Google Scholar]
  44. Russell G. C., Guest J. R. Sequence similarities within the family of dihydrolipoamide acyltransferases and discovery of a previously unidentified fungal enzyme. Biochim Biophys Acta. 1991 Jan 29;1076(2):225–232. doi: 10.1016/0167-4838(91)90271-z. [DOI] [PubMed] [Google Scholar]
  45. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Skinner D. D., Denoya C. D. A simple DNA polymerase chain reaction method to locate and define orientation of specific sequences in cloned bacterial genomic fragments. Microbios. 1993;75(303):125–129. [PubMed] [Google Scholar]
  47. Sokatch J. R., McCully V., Roberts C. M. Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida. J Bacteriol. 1981 Nov;148(2):647–652. doi: 10.1128/jb.148.2.647-652.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sommer R., Tautz D. Minimal homology requirements for PCR primers. Nucleic Acids Res. 1989 Aug 25;17(16):6749–6749. doi: 10.1093/nar/17.16.6749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  50. Suzuki Y., Yamada T. The nucleotide sequence of 16S rRNA gene from Streptomyces lividans TK21. Nucleic Acids Res. 1988 Jan 11;16(1):370–370. doi: 10.1093/nar/16.1.370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sykes P. J., Burns G., Menard J., Hatter K., Sokatch J. R. Molecular cloning of genes encoding branched-chain keto acid dehydrogenase of Pseudomonas putida. J Bacteriol. 1987 Apr;169(4):1619–1625. doi: 10.1128/jb.169.4.1619-1625.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tibbetts M. W., Hafner E. W., Morgenstern M. R., Skinner D. D., Denoya C. D. Cloning of a DNA fragment involved in pigment production in Streptomyces avermitilis. FEMS Microbiol Lett. 1992 Feb 1;70(1):9–13. doi: 10.1016/0378-1097(92)90555-3. [DOI] [PubMed] [Google Scholar]
  53. Wada K., Aota S., Tsuchiya R., Ishibashi F., Gojobori T., Ikemura T. Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Res. 1990 Apr 25;18 (Suppl):2367–2411. doi: 10.1093/nar/18.suppl.2367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wallbrandt P., Tegman V., Jonsson B. H., Wieslander A. Identification and analysis of the genes coding for the putative pyruvate dehydrogenase enzyme complex in Acholeplasma laidlawii. J Bacteriol. 1992 Feb;174(4):1388–1396. doi: 10.1128/jb.174.4.1388-1396.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wang G. F., Kuriki T., Roy K. L., Kaneda T. The primary structure of branched-chain alpha-oxo acid dehydrogenase from Bacillus subtilis and its similarity to other alpha-oxo acid dehydrogenases. Eur J Biochem. 1993 May 1;213(3):1091–1099. doi: 10.1111/j.1432-1033.1993.tb17858.x. [DOI] [PubMed] [Google Scholar]
  56. Wexler I. D., Hemalatha S. G., Patel M. S. Sequence conservation in the alpha and beta subunits of pyruvate dehydrogenase and its similarity to branched-chain alpha-keto acid dehydrogenase. FEBS Lett. 1991 Apr 22;282(1):209–213. doi: 10.1016/0014-5793(91)80479-m. [DOI] [PubMed] [Google Scholar]
  57. Wheelock M. J., Komuniecki R., Duran E., Johnson K. R. Characterization of cDNA clones for the beta subunit of pyruvate dehydrogenase from Ascaris suum. Mol Biochem Parasitol. 1991 Mar;45(1):9–17. doi: 10.1016/0166-6851(91)90022-x. [DOI] [PubMed] [Google Scholar]
  58. Wright F., Bibb M. J. Codon usage in the G+C-rich Streptomyces genome. Gene. 1992 Apr 1;113(1):55–65. doi: 10.1016/0378-1119(92)90669-g. [DOI] [PubMed] [Google Scholar]
  59. Wynn R. M., Chuang J. L., Davie J. R., Fisher C. W., Hale M. A., Cox R. P., Chuang D. T. 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] [Google Scholar]
  60. Yeaman S. J., Hutcheson E. T., Roche T. E., Pettit F. H., Brown J. R., Reed L. J., Watson D. C., Dixon G. H. Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart. Biochemistry. 1978 Jun 13;17(12):2364–2370. doi: 10.1021/bi00605a017. [DOI] [PubMed] [Google Scholar]
  61. Yeaman S. J. The 2-oxo acid dehydrogenase complexes: recent advances. Biochem J. 1989 Feb 1;257(3):625–632. doi: 10.1042/bj2570625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zhang B., Crabb D. W., Harris R. A. Nucleotide and deduced amino acid sequence of the E1 alpha subunit of human liver branched-chain alpha-ketoacid dehydrogenase. Gene. 1988 Sep 15;69(1):159–164. doi: 10.1016/0378-1119(88)90390-3. [DOI] [PubMed] [Google Scholar]
  63. Zhang B., Kuntz M. J., Goodwin G. W., Harris R. A., Crabb D. W. Molecular cloning of a cDNA for the E1 alpha subunit of rat liver branched chain alpha-ketoacid dehydrogenase. J Biol Chem. 1987 Nov 5;262(31):15220–15224. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES