Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 May 15;284(Pt 1):181–187. doi: 10.1042/bj2840181

Differential flux through the quinate and shikimate pathways. Implications for the channelling hypothesis.

H K Lamb 1, J P van den Hombergh 1, G H Newton 1, J D Moore 1, C F Roberts 1, A R Hawkins 1
PMCID: PMC1132714  PMID: 1318019

Abstract

The qutC gene encoding dehydroshikimate dehydratase has been constitutively overexpressed in Aspergillus nidulans from a range of 1-30-fold over the normal wild-type level. This overexpression leads to impaired growth in minimal medium which can be alleviated by the addition of aromatic amino acids to the medium. Overexpression of the qutC gene in mutant strains lacking protocatechuic acid (PCA) oxygenase leads to the build up of PCA in the medium, which can be measured by a simple assay. Measuring the rate of production of PCA in strains overproducing dehydroshikimate dehydratase and correlating this with the level of overproduction and impaired ability to grow in minimal medium lacking aromatic amino acids leads to the conclusion that (a) the metabolites 3-dehydroquinate and dehydroshikimate leak from the AROM protein at a rate comparable with the extent of flux catalysed by the AROM protein, (b) the AROM protein has a low-level channelling function probably as a result of the close juxtaposition of five active sites and (c) this channelling function is only physiologically significant under non-optimal conditions of nutrient supply and oxygenation, when the organism is in situ in its natural environment.

Full text

PDF
181

Selected References

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

  1. Anton I. A., Duncan K., Coggins J. R. A eukaryotic repressor protein, the qa-1S gene product of Neurospora crassa, is homologous to part of the arom multifunctional enzyme. J Mol Biol. 1987 Sep 20;197(2):367–371. doi: 10.1016/0022-2836(87)90130-6. [DOI] [PubMed] [Google Scholar]
  2. Ballance D. J., Buxton F. P., Turner G. Transformation of Aspergillus nidulans by the orotidine-5'-phosphate decarboxylase gene of Neurospora crassa. Biochem Biophys Res Commun. 1983 Apr 15;112(1):284–289. doi: 10.1016/0006-291x(83)91828-4. [DOI] [PubMed] [Google Scholar]
  3. Beri R. K., Grant S., Roberts C. F., Smith M., Hawkins A. R. Selective overexpression of the QUTE gene encoding catabolic 3-dehydroquinase in multicopy transformants of Aspergillus nidulans. Biochem J. 1990 Jan 15;265(2):337–342. doi: 10.1042/bj2650337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beri R. K., Whittington H., Roberts C. F., Hawkins A. R. Isolation and characterization of the positively acting regulatory gene QUTA from Aspergillus nidulans. Nucleic Acids Res. 1987 Oct 12;15(19):7991–8001. doi: 10.1093/nar/15.19.7991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chaleff R. S. The inducible quinate-shikimate catabolic pathway in Neurospora crassa: induction and regulation of enzyme synthesis. J Gen Microbiol. 1974 Apr;81(2):357–372. doi: 10.1099/00221287-81-2-357. [DOI] [PubMed] [Google Scholar]
  6. Charles I. G., Keyte J. W., Brammar W. J., Hawkins A. R. Nucleotide sequence encoding the biosynthetic dehydroquinase function of the penta-functional arom locus of Aspergillus nidulans. Nucleic Acids Res. 1985 Nov 25;13(22):8119–8128. doi: 10.1093/nar/13.22.8119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Charles I. G., Keyte J. W., Brammar W. J., Smith M., Hawkins A. R. The isolation and nucleotide sequence of the complex AROM locus of Aspergillus nidulans. Nucleic Acids Res. 1986 Mar 11;14(5):2201–2213. doi: 10.1093/nar/14.5.2201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clements J. M., Roberts C. F. Molecular cloning of the 3-phosphoglycerate kinase (PGK) gene from Aspergillus nidulans. Curr Genet. 1985;9(4):293–298. doi: 10.1007/BF00419958. [DOI] [PubMed] [Google Scholar]
  9. Coggins J. R., Boocock M. R., Chaudhuri S., Lambert J. M., Lumsden J., Nimmo G. A., Smith D. D. The arom multifunctional enzyme from Neurospora crassa. Methods Enzymol. 1987;142:325–341. doi: 10.1016/s0076-6879(87)42044-2. [DOI] [PubMed] [Google Scholar]
  10. Da Silva A. J., Whittington H., Clements J., Roberts C., Hawkins A. R. Sequence analysis and transformation by the catabolic 3-dehydroquinase (QUTE) gene from Aspergillus nidulans. Biochem J. 1986 Dec 1;240(2):481–488. doi: 10.1042/bj2400481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gaertner F. H., Ericson M. C., DeMoss J. A. Catalytic facilitation in vitro by two multienyzme complexes from Neurospora crassa. J Biol Chem. 1970 Feb 10;245(3):595–600. [PubMed] [Google Scholar]
  12. Grant S., Roberts C. F., Lamb H., Stout M., Hawkins A. R. Genetic regulation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans. J Gen Microbiol. 1988 Feb;134(2):347–358. doi: 10.1099/00221287-134-2-347. [DOI] [PubMed] [Google Scholar]
  13. Hawkins A. R., Francisco Da Silva A. J., Roberts C. F. Cloning and characterization of the three enzyme structural genes QUTB, QUTC and QUTE from the quinic acid utilization gene cluster in Aspergillus nidulans. Curr Genet. 1985;9(4):305–311. doi: 10.1007/BF00419960. [DOI] [PubMed] [Google Scholar]
  14. Hawkins A. R., Francisco da Silva A. J., Roberts C. F. Evidence for two control genes regulating expression of the quinic acid utilization (qut) gene cluster in Aspergillus nidulans. J Gen Microbiol. 1984 Mar;130(3):567–574. doi: 10.1099/00221287-130-3-567. [DOI] [PubMed] [Google Scholar]
  15. Hawkins A. R., Giles N. H., Kinghorn J. R. Genetical and biochemical aspects of quinate breakdown in the filamentous fungus Aspergillus nidulans. Biochem Genet. 1982 Apr;20(3-4):271–286. doi: 10.1007/BF00484424. [DOI] [PubMed] [Google Scholar]
  16. Hawkins A. R., Lamb H. K., Roberts C. F. Structure of the Aspergillus nidulans qut repressor-encoding gene: implications for the regulation of transcription initiation. Gene. 1992 Jan 2;110(1):109–114. doi: 10.1016/0378-1119(92)90452-u. [DOI] [PubMed] [Google Scholar]
  17. Hawkins A. R., Lamb H. K., Smith M., Keyte J. W., Roberts C. F. Molecular organisation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans. Mol Gen Genet. 1988 Oct;214(2):224–231. doi: 10.1007/BF00337715. [DOI] [PubMed] [Google Scholar]
  18. Hawkins A. R., Smith M. Domain structure and interaction within the pentafunctional arom polypeptide. Eur J Biochem. 1991 Mar 28;196(3):717–724. doi: 10.1111/j.1432-1033.1991.tb15870.x. [DOI] [PubMed] [Google Scholar]
  19. Hawkins A. R. The complex Arom locus of Aspergillus nidulans. Evidence for multiple gene fusions and convergent evolution. Curr Genet. 1987;11(6-7):491–498. doi: 10.1007/BF00384611. [DOI] [PubMed] [Google Scholar]
  20. Jeffreys A. J., Wilson V., Wood D., Simons J. P., Kay R. M., Williams J. G. Linkage of adult alpha- and beta-globin genes in X. laevis and gene duplication by tetraploidization. Cell. 1980 Sep;21(2):555–564. doi: 10.1016/0092-8674(80)90493-6. [DOI] [PubMed] [Google Scholar]
  21. Kinghorn J. R., Hawkins A. R. Cloning and expression in Escherichia coli K-12 of the biosynthetic dehydroquinase function of the arom cluster gene from the eucaryote, Aspergillus nidulans. Mol Gen Genet. 1982;186(1):145–152. doi: 10.1007/BF00422927. [DOI] [PubMed] [Google Scholar]
  22. Lamb H. K., Bagshaw C. R., Hawkins A. R. In vivo overproduction of the pentafunctional arom polypeptide in Aspergillus nidulans affects metabolic flux in the quinate pathway. Mol Gen Genet. 1991 Jun;227(2):187–196. doi: 10.1007/BF00259670. [DOI] [PubMed] [Google Scholar]
  23. Lamb H. K., Hawkins A. R., Smith M., Harvey I. J., Brown J., Turner G., Roberts C. F. Spatial and biological characterisation of the complete quinic acid utilisation gene cluster in Aspergillus nidulans. Mol Gen Genet. 1990 Aug;223(1):17–23. doi: 10.1007/BF00315792. [DOI] [PubMed] [Google Scholar]
  24. Lambert J. M., Boocock M. R., Coggins J. R. The 3-dehydroquinate synthase activity of the pentafunctional arom enzyme complex of Neurospora crassa is Zn2+-dependent. Biochem J. 1985 Mar 15;226(3):817–829. doi: 10.1042/bj2260817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tautz D., Renz M. An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem. 1983 Jul 1;132(1):14–19. doi: 10.1016/0003-2697(83)90419-0. [DOI] [PubMed] [Google Scholar]
  26. Welch G. R., Gaertner F. H. Influence of an aggregated multienzyme system on transient time: kinetic evidence for compartmentation by an aromatic-amino-acid synthesizing complex of Neurospora crassa. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4218–4222. doi: 10.1073/pnas.72.11.4218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Whittington H. A., Grant S., Roberts C. F., Lamb H., Hawkins A. R. Identification and isolation of a putative permease gene in the quinic acid utilization (QUT) gene cluster of Aspergillus nidulans. Curr Genet. 1987;12(2):135–139. doi: 10.1007/BF00434668. [DOI] [PubMed] [Google Scholar]
  28. Willetts N. S., Clark A. J., Low B. Genetic location of certain mutations conferring recombination deficiency in Escherichia coli. J Bacteriol. 1969 Jan;97(1):244–249. doi: 10.1128/jb.97.1.244-249.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES