Brassinosteroid-6-oxidases from arabidopsis and tomato catalyze multiple C-6 oxidations in brassinosteroid biosynthesis

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. It has been proposed that BRs are synthesized via t wo parallel pathways, the early and late C-6 oxidation pathways according t o the C-6 oxidation status. The tomato (Lycopersicon esculent um) Dwarf gen e encodes a cytochrome P450 that has be en shown to catalyze the C-6 oxidat ion of 6-deoxocastasterone to castasterone. We isolated an Arabidopsis orth olog (AtBX6ox gene) of the tomato Dwarf gene. The encoded polypeptide has c haracteristics of P450s and is classified into the CYP85 family. The AtBR6o x and tomato Dwarf gene were expressed in yeast and the ability of the tran sformed yeast cells to metabolize 6-deoxo-BRs was tested. Metabolites were analyzed by gas chromatography-mass spectrometry. Both Enzymes catalyze mul tiple steps in BR biosynthesis: 6-deoxoteasterone to teasterone, 3-dehydro- 6-deoxoteasterone to 3-dehydroteasterone, 6-deoxotyphasterol to typhasterol , and 6-deoxocastasterone to castasterone. Our results indicate that the At BR6ox gene and the tomato Dwarf gene encode steroid-6-oxidases and that the se enzymes have a broad substrate specificity. This suggests that the BR bi osynthetic pathway consists of a metabolic grid rather than two separate pa rallel pathways.