Regiospecific hydroxylation of isoflavones by cytochrome p450 81E enzymes from Medicago truncatula.

Mining of Medicago truncatula EST databases and screening of a root cDNA library led to the identification of three cytochrome p450 81E subfamily members. Two were functionally characterized by expression in yeast. The recombinant enzymes in yeast microsomes utilized the same isoflavone substrates, but produced different products hydroxylated at the 2' and/or 3' positions of the B-ring. When transiently expressed in alfalfa leaves, green fluorescent protein (GFP) fusions of the isoflavone 2'- and 3'-hydroxylases localized to the endoplasmic reticulum. The isoflavone 2'-hydroxylase was functional when expressed in Arabidopsis. Differential tissue-specific and biotic/abiotic stress-dependent expression patterns were observed for the isoflavone 2'-hydroxylase and 3'-hydroxylase genes, suggesting differential involvement of 2'- and 3'-hydroxylated isoflavonoids in pathogen defense and insect-induced responses, respectively, in Medicago.