Research article

Purification and kinetic studies of recombinant gibberellin dioxygenases

Diane R Lester¹ , Andy Phillips² , Peter Hedden² and Inger Andersson³

¹Institute for Cell and Molecular Biology, Uppsala University, Box 596, 751 24 Uppsala, Sweden

²Rothamsted Research, Harpenden, Herts, AL5 2JQ UK

³Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, 751 24 Uppsala, Sweden

author email corresponding author email

BMC Plant Biology 2005, 5:19doi:10.1186/1471-2229-5-19

Published:	25 September 2005

Abstract

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 K_m of 0.024 μM and a V_max of 4.4 pkat/mg toward [1β,2β,3β-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 K_m of 0.82 μM and V_max of 52,500 pkat/mg toward [1β,2β,3β-³H₃]GA₂₀.

Conclusion

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.