Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Sep;174(17):5560–5566. doi: 10.1128/jb.174.17.5560-5566.1992

Properties of subcloned subunits of bacterial acetohydroxy acid synthases.

O Weinstock 1, C Sella 1, D M Chipman 1, Z Barak 1
PMCID: PMC206499  PMID: 1512191

Abstract

The acetohydroxy acid synthase (AHAS) isozymes from enterobacteria are each composed of a large and small subunit in an alpha 2 beta 2 structure. It has been generally accepted that the large (ca. 60-kDa) subunits are catalytic, while the small ones are regulatory. In order to further characterize the roles of the subunits as well as the nature and the specificities of their interactions, we have constructed plasmids encoding the large or small subunits of isozymes AHAS I and AHAS III, each with limited remnants of the other peptide. The catalytic properties of the large subunits have been characterized and compared with those of extracts containing the intact enzyme or of purified enzymes. Antisera to the isolated subunits have been used in Western blot (immunoblot) analyses for qualitative and semiquantitative determinations of the presence of the polypeptides in extracts. The large subunits of AHAS isozymes I and III have lower activities than the intact enzymes: Vmax/Km is 20 to 50 times lower in both cases. However, for AHAS I, most of this difference is due to the raised Km of the large subunit alone, while for AHAS III, it is due to a lowered Vmax. The substrate specificities, R, of large subunits are close to those of the intact enzymes. The catalytic activity of the large subunits of AHAS I is dependent on flavin adenine dinucleotide (FAD), as is that of the intact enzyme, although the apparent affinities of the large subunits alone for FAD are 10-fold lower. Isolated subunits are insensitive to valine inhibition. Nearly all of the properties of the intact AHAS isozyme I or III can be reconstituted by mixing extracts containing the respective large and small subunits. The mixing of subunits from different enzymes does not lead to activation of the large subunits. It is concluded that the catalytic machinery of these AHAS isozymes is entirely contained within the large subunits. The small subunits are required, however, for specific stabilization of an active conformation of the large subunits as well as for value sensitivity.

Full text

PDF
5560

Selected References

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

  1. Aulabaugh A., Schloss J. V. Oxalyl hydroxamates as reaction-intermediate analogues for ketol-acid reductoisomerase. Biochemistry. 1990 Mar 20;29(11):2824–2830. doi: 10.1021/bi00463a027. [DOI] [PubMed] [Google Scholar]
  2. Barak Z., Calvo J. M., Schloss J. V. Acetolactate synthase isozyme III from Escherichia coli. Methods Enzymol. 1988;166:455–458. doi: 10.1016/s0076-6879(88)66059-9. [DOI] [PubMed] [Google Scholar]
  3. Barak Z., Chipman D. M., Gollop N. Physiological implications of the specificity of acetohydroxy acid synthase isozymes of enteric bacteria. J Bacteriol. 1987 Aug;169(8):3750–3756. doi: 10.1128/jb.169.8.3750-3756.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. 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]
  6. Chang Y. Y., Cronan J. E., Jr Common ancestry of Escherichia coli pyruvate oxidase and the acetohydroxy acid synthases of the branched-chain amino acid biosynthetic pathway. J Bacteriol. 1988 Sep;170(9):3937–3945. doi: 10.1128/jb.170.9.3937-3945.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dailey F. E., Cronan J. E., Jr Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source. J Bacteriol. 1986 Feb;165(2):453–460. doi: 10.1128/jb.165.2.453-460.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dailey F. E., Cronan J. E., Jr, Maloy S. R. Acetohydroxy acid synthase I is required for isoleucine and valine biosynthesis by Salmonella typhimurium LT2 during growth on acetate or long-chain fatty acids. J Bacteriol. 1987 Feb;169(2):917–919. doi: 10.1128/jb.169.2.917-919.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Eoyang L., Silverman P. M. Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition. J Bacteriol. 1986 Jun;166(3):901–904. doi: 10.1128/jb.166.3.901-904.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gollop N., Damri B., Barak Z., Chipman D. M. Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli. Biochemistry. 1989 Jul 25;28(15):6310–6317. doi: 10.1021/bi00441a024. [DOI] [PubMed] [Google Scholar]
  13. Gollop N., Damri B., Chipman D. M., Barak Z. Physiological implications of the substrate specificities of acetohydroxy acid synthases from varied organisms. J Bacteriol. 1990 Jun;172(6):3444–3449. doi: 10.1128/jb.172.6.3444-3449.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Haughn G. W., Squires C. H., De Felice M., Largo C. T., Calvo J. M. Unusual organization of the ilvIH promoter of Escherichia coli. J Bacteriol. 1985 Jul;163(1):186–198. doi: 10.1128/jb.163.1.186-198.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lago C. T., Sannia G., Marino G., Squires C. H., Calvo J. M., De Felice M. The ilvIH operon of Escherichia coli K-12. Identification of the gene products and recognition of the translational start by polypeptide microsequencing. Biochim Biophys Acta. 1985 Jan 29;824(1):74–79. doi: 10.1016/0167-4781(85)90031-4. [DOI] [PubMed] [Google Scholar]
  17. Lu M. F., Umbarger H. E. Effects of deletion and insertion mutations in the ilvM gene of Escherichia coli. J Bacteriol. 1987 Feb;169(2):600–604. doi: 10.1128/jb.169.2.600-604.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. Sutton A., Freundlich M. Regulation of cyclic AMP of the ilvB-encoded biosynthetic acetohydroxy acid synthase in Escherichia coli K-12. Mol Gen Genet. 1980 Apr;178(1):179–183. doi: 10.1007/BF00267227. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  25. 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]
  26. Yadav N., McDevitt R. E., Benard S., Falco S. C. Single amino acid substitutions in the enzyme acetolactate synthase confer resistance to the herbicide sulfometuron methyl. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4418–4422. doi: 10.1073/pnas.83.12.4418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. de Felice M., Lago C. T., Squires C. H., Calvo J. M. Acetohydroxy acid synthase isoenzymes of Escherichia coli K12 and Salmonella typhimurium. Ann Microbiol (Paris) 1982 Mar-Apr;133(2):251–256. [PubMed] [Google Scholar]

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

RESOURCES