Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1989 Jun 15;260(3):737–747. doi: 10.1042/bj2600737

Overexpression and site-directed mutagenesis of the succinyl-CoA synthetase of Escherichia coli and nucleotide sequence of a gene (g30) that is adjacent to the suc operon.

D Buck 1, J R Guest 1
PMCID: PMC1138739  PMID: 2548486

Abstract

The succinyl-CoA synthetase of Escherichia coli is encoded by two genes, sucC (beta subunit) and sucD (alpha subunit), which are distal genes in the sucABCD operon. They are expressed from the suc promoter, which also expresses the dehydrogenase and dihydrolipoyl succinyl-transferase subunits of the 2-oxoglutarate dehydrogenase complex. Strategies have now been devised for the site-directed mutagenesis and independent expression of the succinyl-CoA synthetase (alpha 2 beta 2 tetramer) and the individual subunits. These involve (1) subcloning a promoterless sucCD fragment downstream of the lac promoter in M13mp10, and (2) precise splicing of the suc coding regions with the efficient atpE ribosome-binding site and expression from the thermoinducible lambda promoters in the pJLA503 vector. Succinyl-CoA synthetase specific activities were amplified 40-60-fold within 5 h of thermoinduction of the lambda promoters, and the alpha and beta subunits accounted for almost 30% of the protein in supernatant fractions of the cell-free extracts. Site-directed mutagenesis of potential CoA binding-site residues indicated that Trp-43 beta and His-50 beta are essential residues in the beta-subunit, whereas Cys-47 beta could be replaced by serine without inactivating the enzyme. No activity was detected after the histidine residue at the phosphorylation site of the alpha-subunit was replaced by aspartate (His-246 alpha----Asp), but this alteration seemed to have a deleterious effect on the accumulation of the enzyme in cell-free supernatant extracts. The nucleotide sequence of an unidentified gene (g30) that is adjacent to the sucABCD operon was defined by extending the sequence of the citric acid cycle gene cluster by 818 bp to 13379 bp: gltA-sdhCDAB-sucABCD-g30. This gene converges on the suc operon and encodes a product (P30) that contains 230 amino acids (Mr 27,251). Highly significant similarities were detected between the N-terminal region of P30 and those of GENA [the product of another unidentified gene (geneA) located upstream of the aceEF-lpd operon], and GNTR (a putative transcriptional repressor of the gluconate operon of Bacillus subtilis). Possible roles for GENA and P30 as transcriptional regulators of the adjacent operons encoding the pyruvate and 2-oxoglutarate dehydrogenase complexes are discussed.

Full text

PDF
737

Images in this article

742Fig. 4.
on p.742

Selected References

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

  1. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bishop M., Thompson E. Fast computer search for similar DNA sequences. Nucleic Acids Res. 1984 Jul 11;12(13):5471–5474. doi: 10.1093/nar/12.13.5471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Buck D., Spencer M. E., Guest J. R. Primary structure of the succinyl-CoA synthetase of Escherichia coli. Biochemistry. 1985 Oct 22;24(22):6245–6252. doi: 10.1021/bi00343a031. [DOI] [PubMed] [Google Scholar]
  5. Collier G. E., Nishimura J. S. Evidence for a second histidine at the active site of succinyl-CoA synthetase from Escherichia coli. J Biol Chem. 1979 Nov 10;254(21):10925–10930. [PubMed] [Google Scholar]
  6. Collins J. F., Coulson A. F., Lyall A. The significance of protein sequence similarities. Comput Appl Biosci. 1988 Mar;4(1):67–71. doi: 10.1093/bioinformatics/4.1.67. [DOI] [PubMed] [Google Scholar]
  7. Darlison M. G., Spencer M. E., Guest J. R. Nucleotide sequence of the sucA gene encoding the 2-oxoglutarate dehydrogenase of Escherichia coli K12. Eur J Biochem. 1984 Jun 1;141(2):351–359. doi: 10.1111/j.1432-1033.1984.tb08199.x. [DOI] [PubMed] [Google Scholar]
  8. Dodd I. B., Egan J. B. Systematic method for the detection of potential lambda Cro-like DNA-binding regions in proteins. J Mol Biol. 1987 Apr 5;194(3):557–564. doi: 10.1016/0022-2836(87)90681-4. [DOI] [PubMed] [Google Scholar]
  9. Fujita Y., Fujita T., Miwa Y., Nihashi J., Aratani Y. Organization and transcription of the gluconate operon, gnt, of Bacillus subtilis. J Biol Chem. 1986 Oct 15;261(29):13744–13753. [PubMed] [Google Scholar]
  10. Grosjean H., Fiers W. Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene. 1982 Jun;18(3):199–209. doi: 10.1016/0378-1119(82)90157-3. [DOI] [PubMed] [Google Scholar]
  11. Henning W. D., Upton C., McFadden G., Majumdar R., Bridger W. A. Cloning and sequencing of the cytoplasmic precursor to the alpha subunit of rat liver mitochondrial succinyl-CoA synthetase. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1432–1436. doi: 10.1073/pnas.85.5.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kramer B., Kramer W., Fritz H. J. Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli. Cell. 1984 Oct;38(3):879–887. doi: 10.1016/0092-8674(84)90283-6. [DOI] [PubMed] [Google Scholar]
  13. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  18. Miles J. S., Guest J. R. Complete nucleotide sequence of the fumarase gene (citG) of Bacillus subtilis 168. Nucleic Acids Res. 1985 Jan 11;13(1):131–140. doi: 10.1093/nar/13.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miles J. S., Guest J. R. Molecular genetic aspects of the citric acid cycle of Escherichia coli. Biochem Soc Symp. 1987;54:45–65. [PubMed] [Google Scholar]
  20. Nishimura J. S. Succinyl-CoA synthetase structure-function relationships and other considerations. Adv Enzymol Relat Areas Mol Biol. 1986;58:141–172. doi: 10.1002/9780470123041.ch4. [DOI] [PubMed] [Google Scholar]
  21. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  22. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  23. Schauder B., Blöcker H., Frank R., McCarthy J. E. Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region. Gene. 1987;52(2-3):279–283. doi: 10.1016/0378-1119(87)90054-0. [DOI] [PubMed] [Google Scholar]
  24. Spencer M. E., Darlison M. G., Stephens P. E., Duckenfield I. K., Guest J. R. Nucleotide sequence of the sucB gene encoding the dihydrolipoamide succinyltransferase of Escherichia coli K12 and homology with the corresponding acetyltransferase. Eur J Biochem. 1984 Jun 1;141(2):361–374. doi: 10.1111/j.1432-1033.1984.tb08200.x. [DOI] [PubMed] [Google Scholar]
  25. Spencer M. E., Guest J. R. Transcription analysis of the sucAB, aceEF and lpd genes of Escherichia coli. Mol Gen Genet. 1985;200(1):145–154. doi: 10.1007/BF00383328. [DOI] [PubMed] [Google Scholar]
  26. Staden R. A new computer method for the storage and manipulation of DNA gel reading data. Nucleic Acids Res. 1980 Aug 25;8(16):3673–3694. doi: 10.1093/nar/8.16.3673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Staden R. A strategy of DNA sequencing employing computer programs. Nucleic Acids Res. 1979 Jun 11;6(7):2601–2610. doi: 10.1093/nar/6.7.2601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Staden R., McLachlan A. D. Codon preference and its use in identifying protein coding regions in long DNA sequences. Nucleic Acids Res. 1982 Jan 11;10(1):141–156. doi: 10.1093/nar/10.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Staden R., McLachlan A. D. Codon preference and its use in identifying protein coding regions in long DNA sequences. Nucleic Acids Res. 1982 Jan 11;10(1):141–156. doi: 10.1093/nar/10.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stephens P. E., Darlison M. G., Lewis H. M., Guest J. R. 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. doi: 10.1111/j.1432-1033.1983.tb07441.x. [DOI] [PubMed] [Google Scholar]
  31. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  32. Weitzman P. D. Unity and diversity in some bacterial citric acid-cycle enzymes. Adv Microb Physiol. 1981;22:185–244. doi: 10.1016/s0065-2911(08)60328-8. [DOI] [PubMed] [Google Scholar]
  33. Wolodko W. T., Brownie E. R., Bridger W. A. Subunits of succinyl-coenzyme A synthetase: coordination of production in Escherichia coli and discovery of a factor that precludes refolding. J Bacteriol. 1980 Jul;143(1):231–237. doi: 10.1128/jb.143.1.231-237.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wolodko W. T., James M. N., Bridger W. A. Crystallization of succinyl-CoA synthetase from Escherichia coli. J Biol Chem. 1984 Apr 25;259(8):5316–5320. [PubMed] [Google Scholar]
  35. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  36. Ybarra J., Prasad A. R., Nishimura J. S. Chemical modification of tryptophan residues in Escherichia coli succinyl-CoA synthetase. Effect on structure and enzyme activity. Biochemistry. 1986 Nov 4;25(22):7174–7178. doi: 10.1021/bi00370a061. [DOI] [PubMed] [Google Scholar]
  37. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]
  38. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES