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.
Liu
Chang-Jun
CJ
Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA.
Huhman
David
D
Sumner
Lloyd W
LW
Dixon
Richard A
RA
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins; D006900:Hydroxylation; D007529:Isoflavones / Q000096:biosynthesis*; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D029902:Medicago / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D007529:Isoflavones; D008164:Luminescent Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C115412:cytochrome P-450 CYP81E1 (Glycyrrhiza echinata); C483421:cytochrome P-450 CYP81E7, Medicago truncatula; C483422:cytochrome P-450 CYP81E8, Medicago truncatula; C483423:cytochrome P-450 CYP81E9, Medicago truncatula