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. 1994 Aug 30;91(18):8552–8556. doi: 10.1073/pnas.91.18.8552

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

T Lange 1, P Hedden 1, J E Graebe 1
PMCID: PMC44644  PMID: 8078921

Abstract

In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA25 and of GA53 to GA17, as well as the formation of the C19-GAs, GA1, GA9, and GA20, from their respective aldehyde precursors, GA23, GA24, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted M(r) (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases.

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Selected References

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  1. Britsch L., Dedio J., Saedler H., Forkmann G. Molecular characterization of flavanone 3 beta-hydroxylases. Consensus sequence, comparison with related enzymes and the role of conserved histidine residues. Eur J Biochem. 1993 Oct 15;217(2):745–754. doi: 10.1111/j.1432-1033.1993.tb18301.x. [DOI] [PubMed] [Google Scholar]
  2. Britsch L., Ruhnau-Brich B., Forkmann G. Molecular cloning, sequence analysis, and in vitro expression of flavanone 3 beta-hydroxylase from Petunia hybrida. J Biol Chem. 1992 Mar 15;267(8):5380–5387. [PubMed] [Google Scholar]
  3. Deikman J., Fischer R. L. Interaction of a DNA binding factor with the 5'-flanking region of an ethylene-responsive fruit ripening gene from tomato. EMBO J. 1988 Nov;7(11):3315–3320. doi: 10.1002/j.1460-2075.1988.tb03202.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gilmour S. J., Zeevaart J. A., Schwenen L., Graebe J. E. Gibberellin metabolism in cell-free extracts from spinach leaves in relation to photoperiod. Plant Physiol. 1986 Sep;82(1):190–195. doi: 10.1104/pp.82.1.190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hedden P. 2-Oxoglutarate-dependent dioxygenases in plants: mechanism and function. Biochem Soc Trans. 1992 May;20(2):373–377. doi: 10.1042/bst0200373. [DOI] [PubMed] [Google Scholar]
  6. Matsuda J., Okabe S., Hashimoto T., Yamada Y. Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger. J Biol Chem. 1991 May 25;266(15):9460–9464. [PubMed] [Google Scholar]
  7. Menssen A., Höhmann S., Martin W., Schnable P. S., Peterson P. A., Saedler H., Gierl A. The En/Spm transposable element of Zea mays contains splice sites at the termini generating a novel intron from a dSpm element in the A2 gene. EMBO J. 1990 Oct;9(10):3051–3057. doi: 10.1002/j.1460-2075.1990.tb07501.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Myllylä R., Günzler V., Kivirikko K. I., Kaska D. D. Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases. Biochem J. 1992 Sep 15;286(Pt 3):923–927. doi: 10.1042/bj2860923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Spanu P., Reinhardt D., Boller T. Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its mRNA in Xenopus laevis oocytes. EMBO J. 1991 Aug;10(8):2007–2013. doi: 10.1002/j.1460-2075.1991.tb07730.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Szoka P. R., Schreiber A. B., Chan H., Murthy J. A general method for retrieving the components of a genetically engineered fusion protein. DNA. 1986 Feb;5(1):11–20. doi: 10.1089/dna.1986.5.11. [DOI] [PubMed] [Google Scholar]

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