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Separation and characterisation of three 2-oxoglutarate-dependent dioxygenases from Cucurbita maxima L. endosperm involved in gibberellin biosynthesis

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Abstract

Three enzymes of the gibberellin (GA) biosynthetic pathway, a 7-oxidase, a 20-oxidase and a 3β-hydroxylase, were partially purified from Cucurbita maxima endosperm by ammonium sulfate precipitation, gel-filtration and anion-exchange chromatography. The enzyme activities, which were assayed by the oxidation of GA12-aldehyde to GA12, of GA12 to GA15 (and GA24) and of GA15 to GA37, respectively, were completely separated from each other. The apparent molecular masses as estimated by gel-filtration high-performance liquid chromatography were 34.5 kDa for the 7-oxidase, 44.5 kDa for the 20-oxidase and 58 kDa for the 3β-hydroxylase. The Michaelis-Menten constants (K m) were 8.6 μM, 0.15μM and 8.7 μM for the respective substrates. All three enzymes had properties typical of 2-oxoglutarate dependent dioxygenases. 2-Oxoglutarate was essential for activity and served as a co-substrate, giving K m values of 6.1 μM, 91 μM and 41 μM with the 7-oxidase, 20-oxidase and 3β-hydroxylase, respectively. Furthermore, 2 oxo[5-14C]glutarate was oxidised stoichiometrically to [14C]succinate when the GA-substrates were oxidised to their respective products, and the 1∶1 ratio was maintained under different oxygen concentrations. Approximately equimolar amounts of 14CO2 were released from 2-oxo[1-14C]glutarate when GA12 was oxidised to GA15/24 by the 20-oxidase. A crude enzyme preparation containing all three enzyme activities (and a 2β-hydroxylase) converted GA12-aldehyde to [18O2]GA4 and [18O5]GA43 under 18O2, showing that all O-atoms incorporated after GA12-aldehyde originate from O2. Accordingly, the reaction rates were near zero under anaerobic conditions, although very low concentrations of O2 sufficed to sustain the reactions. Both Fe2+ and dithiothreitol stimulated the enzyme activities strongly, but if they were added together, catalase was needed to prevent inhibition. The pH dependence showed two opposite trends; the 7-oxidase was most active at pH 6 and below, whereas the other enzymes were maximally active above pH 6.5.

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Abbreviations

BSA:

bovine serum albumin

GAn :

gibberellin An

DTT:

dithiothreitol

GC-MS:

combined gas chromatography-mass spectrometry

MeTMSi:

methyl ester trimethylsilyl ether

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Authors and Affiliations

  1. Pflanzenphysiologisches Institut und Botanischer Garten der Universität Göttingen, Untere Karspüle 2, D-37073, Göttingen, Germany

    Theodor Lange, Antje Schweimer & Jan E. Graebe

  2. Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, BS18 9AF, Bristol, UK

    Dennis A. Ward & Peter Hedden

Authors
  1. Theodor Lange
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  2. Antje Schweimer
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  3. Dennis A. Ward
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  4. Peter Hedden
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  5. Jan E. Graebe
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Additional information

We thank Mr. Keith Hall (Long Ashton) for assistance with the oxygen concentration measurements and Mrs. Gudrun Bodtke (Göttingen) and Mrs. Brigitte Schattenberg (Göttingen) for able technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft, Germany, and the Agricultural and Food Research Council, UK, and by an Academic Research Collaboration award jointly from the Deutsche Akademische Austauschdienst (DAAD) and the British Council.

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Lange, T., Schweimer, A., Ward, D.A. et al. Separation and characterisation of three 2-oxoglutarate-dependent dioxygenases from Cucurbita maxima L. endosperm involved in gibberellin biosynthesis. Planta 195, 98–107 (1994). https://doi.org/10.1007/BF00206297

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