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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1996 Mar 5;93(5):1962–1966. doi: 10.1073/pnas.93.5.1962

Single-amino acid substitutions eliminate lysine inhibition of maize dihydrodipicolinate synthase.

J M Shaver 1, D C Bittel 1, J M Sellner 1, D A Frisch 1, D A Somers 1, B G Gengenbach 1
PMCID: PMC39891  PMID: 8700867

Abstract

Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) catalyzes the first step in biosynthesis of lysine in plants and bacteria. DHPS in plants is highly sensitive to end-product inhibition by lysine and, therefore, has an important role in regulating metabolite flux into lysine. To better understand the feedback inhibition properties of the plant enzyme, we transformed a maize cDNA for lysine-sensitive DHPS into an Escherichia coli strain lacking DHPS activity. Cells were mutagenized with ethylmethanesulfonate, and potential DHPS mutants were selected by growth on minimal medium containing the inhibitory lysine analogue S-2-aminoethyl-L-cysteine. DHPS assays identified surviving colonies expressing lysine-insensitive DHPS activity. Ten single-base-pair mutations were identified in the maize DHPS cDNA sequence; these mutations were specific to one of three amino acid residues (amino acids 157, 162, and 166) localized within a short region of the polypeptide. No other mutations were present in the remaining DHPS cDNA sequence, indicating that altering only one of the three residues suffices to eliminate lysine inhibition of maize DHPS. Identification of these specific mutations that change the highly sensitive maize DHPS to a lysine-insensitive isoform will help resolve the lysine-binding mechanism and the resultant conformational changes involved in inhibition of DHPS activity. The plant-derived mutant DHPS genes may also be used to improve nutritional quality of maize or other cereal grains that have inadequate lysine content when fed to animals such as poultry, swine, or humans.

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Selected References

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  1. Bonnassie S., Oreglia J., Sicard A. M. Nucleotide sequence of the dapA gene from Corynebacterium glutamicum. Nucleic Acids Res. 1990 Nov 11;18(21):6421–6421. doi: 10.1093/nar/18.21.6421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brochetto-Braga M. R., Leite A., Arruda P. Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms. Plant Physiol. 1992 Mar;98(3):1139–1147. doi: 10.1104/pp.98.3.1139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown A. P., Coleman J., Tommey A. M., Watson M. D., Slabas A. R. Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases. Plant Mol Biol. 1994 Oct;26(1):211–223. doi: 10.1007/BF00039533. [DOI] [PubMed] [Google Scholar]
  4. Delauney A. J., Verma D. P. A soybean gene encoding delta 1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated. Mol Gen Genet. 1990 May;221(3):299–305. doi: 10.1007/BF00259392. [DOI] [PubMed] [Google Scholar]
  5. Dereppe C., Bold G., Ghisalba O., Ebert E., Schär H. P. Purification and characterization of dihydrodipicolinate synthase from pea. Plant Physiol. 1992 Mar;98(3):813–821. doi: 10.1104/pp.98.3.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Falco S. C., Guida T., Locke M., Mauvais J., Sanders C., Ward R. T., Webber P. Transgenic canola and soybean seeds with increased lysine. Biotechnology (N Y) 1995 Jun;13(6):577–582. doi: 10.1038/nbt0695-577. [DOI] [PubMed] [Google Scholar]
  7. Frisch D. A., Gengenbach B. G., Tommey A. M., Sellner J. M., Somers D. A., Myers D. E. Isolation and characterization of dihydrodipicolinate synthase from maize. Plant Physiol. 1991 Jun;96(2):444–452. doi: 10.1104/pp.96.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Frisch D. A., Tommey A. M., Gengenbach B. G., Somers D. A. Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA- auxotroph. Mol Gen Genet. 1991 Aug;228(1-2):287–293. doi: 10.1007/BF00282478. [DOI] [PubMed] [Google Scholar]
  9. Gantt J. S., Larson R. J., Farnham M. W., Pathirana S. M., Miller S. S., Vance C. P. Aspartate aminotransferase in effective and ineffective alfalfa nodules : cloning of a cDNA and determination of enzyme activity, protein, and mRNA levels. Plant Physiol. 1992 Mar;98(3):868–878. doi: 10.1104/pp.98.3.868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kaneko T., Hashimoto T., Kumpaisal R., Yamada Y. Molecular cloning of wheat dihydrodipicolinate synthase. J Biol Chem. 1990 Oct 15;265(29):17451–17455. [PubMed] [Google Scholar]
  11. Karchi H., Shaul O., Galili G. Lysine synthesis and catabolism are coordinately regulated during tobacco seed development. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2577–2581. doi: 10.1073/pnas.91.7.2577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kumpaisal R., Hashimoto T., Yamada Y. Purification and characterization of dihydrodipicolinate synthase from wheat suspension cultures. Plant Physiol. 1987 Sep;85(1):145–151. doi: 10.1104/pp.85.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laber B., Gomis-Rüth F. X., Romão M. J., Huber R. Escherichia coli dihydrodipicolinate synthase. Identification of the active site and crystallization. Biochem J. 1992 Dec 1;288(Pt 2):691–695. doi: 10.1042/bj2880691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mirwaldt C., Korndörfer I., Huber R. The crystal structure of dihydrodipicolinate synthase from Escherichia coli at 2.5 A resolution. J Mol Biol. 1995 Feb 10;246(1):227–239. doi: 10.1006/jmbi.1994.0078. [DOI] [PubMed] [Google Scholar]
  15. Perl A., Shaul O., Galili G. Regulation of lysine synthesis in transgenic potato plants expressing a bacterial dihydrodipicolinate synthase in their chloroplasts. Plant Mol Biol. 1992 Aug;19(5):815–823. doi: 10.1007/BF00027077. [DOI] [PubMed] [Google Scholar]
  16. Pisabarro A., Malumbres M., Mateos L. M., Oguiza J. A., Martín J. F. A cluster of three genes (dapA, orf2, and dapB) of Brevibacterium lactofermentum encodes dihydrodipicolinate synthase, dihydrodipicolinate reductase, and a third polypeptide of unknown function. J Bacteriol. 1993 May;175(9):2743–2749. doi: 10.1128/jb.175.9.2743-2749.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Richaud F., Richaud C., Ratet P., Patte J. C. Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene. J Bacteriol. 1986 Apr;166(1):297–300. doi: 10.1128/jb.166.1.297-300.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Saito K., Miura N., Yamazaki M., Hirano H., Murakoshi I. Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8078–8082. doi: 10.1073/pnas.89.17.8078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shaul O., Galili G. Concerted regulation of lysine and threonine synthesis in tobacco plants expressing bacterial feedback-insensitive aspartate kinase and dihydrodipicolinate synthase. Plant Mol Biol. 1993 Nov;23(4):759–768. doi: 10.1007/BF00021531. [DOI] [PubMed] [Google Scholar]
  20. Silk G. W., Matthews B. F., Somers D. A., Gengenbach B. G. Cloning and expression of the soybean DapA gene encoding dihydrodipicolinate synthase. Plant Mol Biol. 1994 Nov;26(3):989–993. doi: 10.1007/BF00028865. [DOI] [PubMed] [Google Scholar]
  21. Snustad D. P., Hunsperger J. P., Chereskin B. M., Messing J. Maize glutamine synthetase cDNAs: isolation by direct genetic selection in Escherichia coli. Genetics. 1988 Dec;120(4):1111–1123. doi: 10.1093/genetics/120.4.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Van Camp W., Bowler C., Villarroel R., Tsang E. W., Van Montagu M., Inzé D. Characterization of iron superoxide dismutase cDNAs from plants obtained by genetic complementation in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9903–9907. doi: 10.1073/pnas.87.24.9903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. YUGARI Y., GILVARG C. Coordinate end-product inhibition in lysine synthesis in Escherichia coli. Biochim Biophys Acta. 1962 Aug 27;62:612–614. doi: 10.1016/0006-3002(62)90256-1. [DOI] [PubMed] [Google Scholar]

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