Publications

• Purification and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima L. endosperm.

  Lange T.

  Gibberellin (GA) 20-oxidase was purified to apparent homogeneity from Cucurbita maxima endosperm by fractionated ammonium-sulphate precipitation, gel-filtration chromatography and anion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Average purification after t ... >> More

  Gibberellin (GA) 20-oxidase was purified to apparent homogeneity from Cucurbita maxima endosperm by fractionated ammonium-sulphate precipitation, gel-filtration chromatography and anion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Average purification after the last step was 55-fold with 3.9% of the activity recovered. The purest single fraction was enriched 101-fold with 0.2% overall recovery. Apparent relative molecular mass of the enzyme was 45 kDa, as determined by gel-filtration HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, indicating that GA 20-oxidase is probably a monomeric enzyme. The purified enzyme degraded on two-dimensional gel electrophoresis, giving two protein spots: a major one corresponding to a molecular mass of 30 kDa and a minor one at 45 kDa. The isoelectric point for both was 5.4. The amino-acid sequences of the amino-terminus of the purified enzyme and of two peptides from a tryptic digest were determined. The purified enzyme catalysed the sequential conversion of [14C]GA12 to [14C]GA15, [14C]GA24 and [14C]GA25, showing that carbon atom 20 was oxidised to the corresponding alcohol, aldehyde and carboxylic acid in three consecutive reactions. [14C]Gibberellin A53 was similarly converted to [14C]GA44, [14C]GA19, [14C]GA17 and small amounts of a fourth product, which was preliminarily identified as [14C]GA20, a C19-gibberellin. All GAs except [14C]GA20 were identified by combined gas chromatography-mass spectrometry. The cofactor requirements in the absence of dithiothreitol were essentially as in its presence (Lange et al., Planta 195, 98-107, 1994), except that ascorbate was essential for enzyme activity and the optimal concentration of catalase was lower. << Less

  Planta 195:108-115(1994) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli.

  Lange T.

  Gibberellin (GA) plant hormones are biosynthesized via complex pathways, the final steps of which are catalyzed by 2-oxoglutarate-dependent dioxygenases. Here, the cloning of two such enzymes, the GA 7-oxidase and the GA 20-oxidase, is reported using a novel approach, namely, by screening for GA d ... >> More

  Gibberellin (GA) plant hormones are biosynthesized via complex pathways, the final steps of which are catalyzed by 2-oxoglutarate-dependent dioxygenases. Here, the cloning of two such enzymes, the GA 7-oxidase and the GA 20-oxidase, is reported using a novel approach, namely, by screening for GA dioxygenase activities expressed as T7 gene 10 fusion proteins in recombinant Escherichia coli. In vitro translation products of mRNA from endosperm of immature pumpkin seeds contained three GA dioxygenase activities, including 7-oxidase, 20-oxidase, and 3beta-hydroxylase. A cDNA expression library was prepared from the endosperm mRNA in lambdaMOSElox. An aliquot of the amplified library was converted to plasmids in vivo and used for transformation of E. coli BL21(DE3), which thereafter expressed recombinant fusion proteins containing 7-oxidase, 20-oxidase, and 3beta-hydroxylase activities. By screening for specific GA dioxygenase expression, clones harboring 7-oxidase and 20-oxidase cDNA were isolated. The ORF of the 7-oxidase cDNA is 945 bp long, encoding for 314 amino acid residues with a calculated Mr of 35,712 and pI of 5.7. Recombinant GA 7-oxidase oxidizes GA12-aldehyde to GA12 and GA14-aldehyde to GA14. Evidence was obtained for further metabolism of GA12 by the 7-oxidase to four products, two of which are monohydroxylated GA12. The ORF of the 20-oxidase is-apart from seven changes, resulting in four amino acid substitutions-identical to the 20-oxidase cDNA previously cloned from pumpkin cotyledon mRNA; both 20-oxidases have the same catalytic properties. << Less

  Proc Natl Acad Sci U S A 94:6553-6558(1997) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis.

  Phillips A.L., Ward D.A., Uknes S., Appleford N.E.J., Lange T., Huttly A.K., Gaskin P., Graebe J.E., Hedden P.

  Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (GA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from arabidopsis thaliana genomic DNA. One of these was used to isolate two different full-l ... >> More

  Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (GA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from arabidopsis thaliana genomic DNA. One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the GA-deficient Arabidopsis mutant ga1-2. A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags. The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized GA12 at C-20 to GA15, GA24, and the C19 compound GA9, a precursor of bioactive GAs; the C20 tricarboxylic acid compound GA25 was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate GA53, but less effectively than GA12. The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing siliques, and YAP169 in siliques only. In the floral shoots of the ga1-2 mutant, transcript levels corresponding to each cDNA decreased dramatically after GA3 application, suggesting that GA biosynthesis may be controlled, at least in part, through down-regulation of the expression of the 20-oxidase genes. << Less

  Plant Physiol. 108:1049-1057(1995) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.