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Structure and function of gibberellin-biosynthetic enzymes

Reference	BBS/E/C/00803286
Principal Investigator / Supervisor	Professor Peter Hedden
Co-Investigators / Co-Supervisors
Institution	Rothamsted Research
Department	Rothamsted Research Department
Funding type	Research
Value (£)	296,842
Status	Completed
Type	Institute Project
Start date	01/04/1997
End date	31/03/1999
Duration	24 months

Abstract

Gibberellins (GAs) are endogenous plant growth hormones that control many developmental processes in higher plants, including stem extension, flower development, fruit growth and seed germination. These hormones are diterpenoids which are produced by an unusually complex biosynthetic pathway. In order to understand the metabolic control of GA concentration, it is necessary to characterise fully the enzymes involved in GA biosynthesis, to define precisely the reactions they catalyse and to identify factors that influence their activity. There is particular emphasis on the 2-oxoglutarate- dependent dioxygenases which catalyse the later steps of the pathway. Native enzymes purified from developing seeds and recombinant enzymes from heterologous expression of cDNAs are being studied. Reaction mechanisms are being investigated using isotopically labelled substrate analogues in combination with mass spectrometry and nuclear magnetic resonance spectroscopy. Enzyme structure will be determined by affinity labelling and X- ray crystallography. Objectives 1996 Conduct steady state kinetics on GA12 20-oxidation using one or more of the recombinant Arabidopsis GA 20-oxidases prepared by over-expression in E. coli. Prepare sufficient stable recombinant GA 20- oxidase for X-ray crystallisation studies by expressing a thioredoxin - 20-oxidase fusion protein in E. coli using the pTrxfus vector. Photoaffinity label recombinant GA 20- oxidase using reagents based on GA12. Prepare GA 20-homo analogues and study their metabolism by recombinant GA 20-oxidases. 1997 The stoichiometry for the conversion of GA24 to GA9 and of 2-oxglutarate to succinate will be determined with recombinant GA 20-oxidase in order to acertain the number of oxidative steps required for loss of C- 20. 19- HomoGA12 analogues will be prepared and their metabolism by recombinant GA 20-oxidase determined. Radiolabelled photoaffinity reagents based on GA12 and GA9 will be prepared and used to label the active sites of the 20- oxidase (GA12) and 3beta-hydroxylase (GA9). Proteins labelled with these photoaffinity reagents and with an acylcyclohexanedione-based reagent will be examined by LC-MS after limited proteolysis. The number of enzymes involved in the conversion of GSA9 to GA4 and GA7 in embryos of Marah macrocarpus will be determined by enzyme purification.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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