Function and substrate specificity of the gibberellin 3beta-hydroxylase encoded by the Arabidopsis GA4 gene.
cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh. ) was expressed in Escherichia coli, from which cell lysates converted [14C]gibberellin (GA)9 and [14C]GA20 to radiolabeled GA4 and GA1, respectively, thereby confirming that GA4 encodes a GA 3beta-hydroxylase. GA9 was the preferred substrate, with a Michaelis value of 1 microm compared with 15 microm for GA20. Hydroxylation of these GAs was regiospecific, with no indication of 2beta-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA12-aldehyde, GA12, GA19, GA25, GA53, or GA44 as the open lactone (20-hydroxy-GA53), whereas GA15, GA24, and GA44 were hydroxylated to GA37, GA36, and GA38, respectively. The open lactone of GA15 (20-hydroxy-GA12) was hydroxylated but less efficiently than GA15. In contrast to the free acid, GA25 19,20-anhydride was 3beta-hydroxylated to give GA13. 2,3-Didehydro-GA9 and GA5 were converted by recombinant GA4 to the corresponding epoxides 2, 3-oxido-GA9 and GA6.
Williams
J
J
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, United Kingdom.
Phillips
A L
AL
Gaskin
P
P
Hedden
P
P
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D017343:Genes, Plant; D005875:Gibberellins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D005875:Gibberellins; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C108186:gibberellin 3beta-hydroxylase