Purification and kinetic studies of recombinant gibberellin dioxygenases.
BACKGROUND
The 2-oxoglutarate-dependent dioxygenases (2ODDs) of gibberellin (GA) biosynthesis have a key role in the metabolism of a major plant hormone. The activity of recombinant GA 2ODDs from many species has been characterised in detail, however little information relates to enzyme purification. Native GA 2ODDs displayed lability during purification.


RESULTS
Two GA 2ODDs were expressed in Escherichia coli and purified to homogeneity. The GA 2-oxidase from Pisum sativum L., PsGA2OX1, was expressed as a glutathione s-transferase (GST) fusion. It was purified in the three steps of affinity chromatography, GST removal and gel filtration. Highly pure PsGA2OX1 was obtained at a yield of 0.3 mg/g of cells. It displayed a Km of 0.024 microM and a Vmax of 4.4 pkat/mg toward [1beta,2beta,3beta-3H3]GA20. The GA 3-oxidase from Arabidopsis thaliana, AtGA3OX4, was expressed as a poly(His)-tagged thioredoxin fusion. It was purified by Immobilised Metal Affinity Chromatography followed by gel filtration. Cleavage of the fusion took place between the two purification steps. Highly pure AtGA3OX4 was obtained at a yield of 0.01 mg/g of cells. It displayed a Km of 0.82 microM and Vmax of 52,500 pkat/mg toward [1beta,2beta,3beta-3H3]GA20.


CONCLUSIONS
Fusion tags were required to stabilise and solubilise PsGA2OX1 and AtGA3OX4 during E. coli expression. The successful purification of milligram quantities of PsGA2OX1 enables mechanistic and structural studies not previously possible on GA 2ODDs. A moderate yield of pure AtGA3OX4 requires the further optimisation of the latter stages of the enzyme purification schedule. PsGA2OX1's action in planta as deduced from the effect of the null mutation sln on GA levels in seeds is in agreement with the kinetic parameters of the recombinant enzyme.
Lester
Diane R
DR
Institute for Cell and Molecular Biology, Uppsala University, 751 24 Uppsala, Sweden. diane@xray.bmc.uu.se
Phillips
Andy
A
Hedden
Peter
P
Andersson
Inger
I
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D006899:Mixed Function Oxygenases; C121463:gibberellin 2-dioxygenase; C108186:gibberellin 3beta-hydroxylase; D005982:Glutathione Transferase