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. 1986 Jun;166(3):901–904. doi: 10.1128/jb.166.3.901-904.1986

Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition.

L Eoyang, P M Silverman
PMCID: PMC215211  PMID: 3011751

Abstract

Most of the coding sequence for the IlvN polypeptide subunit of acetohydroxyacid synthase I was deleted from the ilvB+ ilvN+ plasmid pTCN12 by in vitro methods. Several ilvB+ delta ilvN derivatives of pTCN12 were identified among transformants of a strain otherwise lacking any acetohydroxyacid synthase. Deletion derivatives produced an enzymatically active IlvB polypeptide, as shown by the Ilv+ phenotype of transformed cells and by immunologic and enzymatic assays. However, whereas the growth of pTCN12 transformants was sensitive to valine inhibition, growth of the ilvB+ delta ilvN transformants was relatively resistant. Moreover, in vitro analyses confirmed that both acetolactate and acetohydroxybutyrate synthesis in extracts of the ilvB+ delta ilvN transformants was resistant to valine inhibition, in comparison with that in extracts of pTCN12 transformants or with that catalyzed by purified acetohydroxyacid synthase I. The IlvN polypeptide had a minimal effect, if any, on IlvB polypeptide accumulation as measured by immunoprecipitation, but its absence resulted in a greater than 10-fold reduction in enzyme specific activity.

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Selected References

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

  1. Albin R., Silverman P. M. Physical and genetic structure of the glpK-cpxA interval of the Escherichia coli K-12 chromosome. Mol Gen Genet. 1984;197(2):261–271. doi: 10.1007/BF00330972. [DOI] [PubMed] [Google Scholar]
  2. Albin R., Weber R., Silverman P. M. The Cpx proteins of Escherichia coli K12. Immunologic detection of the chromosomal cpxA gene product. J Biol Chem. 1986 Apr 5;261(10):4698–4705. [PubMed] [Google Scholar]
  3. Calhoun D. H., Rimerman R. A., Hatfield G. W. Threonine deaminase from Escherichia coli. I. Purification and properties. J Biol Chem. 1973 May 25;248(10):3511–3516. [PubMed] [Google Scholar]
  4. De Felice M., Guardiola J., Esposito B., Iaccarino M. Structural genes for a newly recognized acetolactate synthase in Escherichia coli K-12. J Bacteriol. 1974 Dec;120(3):1068–1077. doi: 10.1128/jb.120.3.1068-1077.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eoyang L., Silverman P. M. Purification and subunit composition of acetohydroxyacid synthase I from Escherichia coli K-12. J Bacteriol. 1984 Jan;157(1):184–189. doi: 10.1128/jb.157.1.184-189.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Friden P., Donegan J., Mullen J., Tsui P., Freundlich M., Eoyang L., Weber R., Silverman P. M. The ilvB locus of Escherichia coli K-12 is an operon encoding both subunits of acetohydroxyacid synthase I. Nucleic Acids Res. 1985 Jun 11;13(11):3979–3993. doi: 10.1093/nar/13.11.3979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Guardiola J., De Felice M., Lamberti A., Iaccarino M. The acetolactate synthase isoenzymes of Escherichia coli K-12. Mol Gen Genet. 1977 Nov 4;156(1):17–25. doi: 10.1007/BF00272247. [DOI] [PubMed] [Google Scholar]
  8. McEwen J., Silverman P. Mutations in genes cpxA and cpxB of Escherichia coli K-12 cause a defect in isoleucine and valine syntheses. J Bacteriol. 1980 Oct;144(1):68–73. doi: 10.1128/jb.144.1.68-73.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Newman T. C., Levinthal M. A new map location for the ilvB locus of Escherichia coli. Genetics. 1980 Sep;96(1):59–77. doi: 10.1093/genetics/96.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Newman T., Friden P., Sutton A., Freundlich M. Cloning and expression of the ilvB gene of Escherichia coli K-12. Mol Gen Genet. 1982;186(3):378–384. doi: 10.1007/BF00729457. [DOI] [PubMed] [Google Scholar]
  11. Schloss J. V., Van Dyk D. E., Vasta J. F., Kutny R. M. Purification and properties of Salmonella typhimurium acetolactate synthase isozyme II from Escherichia coli HB101/pDU9. Biochemistry. 1985 Aug 27;24(18):4952–4959. doi: 10.1021/bi00339a034. [DOI] [PubMed] [Google Scholar]
  12. Shaw K. J., Berg C. M. Radioenzymatic assay for the acetohydroxy acid synthase-catalyzed synthesis of alpha-aceto-alpha-hydroxybutyrate. Anal Biochem. 1980 Jun;105(1):101–105. doi: 10.1016/0003-2697(80)90429-7. [DOI] [PubMed] [Google Scholar]
  13. Squires C. H., De Felice M., Devereux J., Calvo J. M. Molecular structure of ilvIH and its evolutionary relationship to ilvG in Escherichia coli K12. Nucleic Acids Res. 1983 Aug 11;11(15):5299–5313. doi: 10.1093/nar/11.15.5299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Squires C. H., De Felice M., Lago C. T., Calvo J. M. IlvHI locus of Salmonella typhimurium. J Bacteriol. 1983 Jun;154(3):1054–1063. doi: 10.1128/jb.154.3.1054-1063.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Squires C. H., De Felice M., Wessler S. R., Calvo J. M. Physical characterization of the ilvHI operon of Escherichia coli K-12. J Bacteriol. 1981 Sep;147(3):797–804. doi: 10.1128/jb.147.3.797-804.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Umbarger H. E. Amino acid biosynthesis and its regulation. Annu Rev Biochem. 1978;47:532–606. doi: 10.1146/annurev.bi.47.070178.002533. [DOI] [PubMed] [Google Scholar]
  17. Wek R. C., Hauser C. A., Hatfield G. W. The nucleotide sequence of the ilvBN operon of Escherichia coli: sequence homologies of the acetohydroxy acid synthase isozymes. Nucleic Acids Res. 1985 Jun 11;13(11):3995–4010. doi: 10.1093/nar/13.11.3995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Whittier R. F., Chase J. W. DNA repair in E. coli strains deficient in single-strand DNA binding protein. Mol Gen Genet. 1981;183(2):341–347. doi: 10.1007/BF00270638. [DOI] [PubMed] [Google Scholar]

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