Regiospecific hydroxylation of isoflavones by cytochrome P450 81E enzymes from Medicago truncatula

• Published in: The Plant journal : for cell and molecular biology Vol. 36; no. 4; pp. 471 - 484
• Main Authors: Liu, Chang‐Jun, Huhman, David, Sumner, Lloyd W., Dixon, Richard A.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.11.2003; Blackwell Science
• Subjects: Amino Acid Sequence; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis thaliana; Biological and medical sciences; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450; elicitation; Expressed sequence tags; functional expression; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Green Fluorescent Proteins; Hydroxylation; insect herbivory; isoflavone 2'-hydroxylase; isoflavone 3'-hydroxylase; Isoflavones - biosynthesis; isoflavonoid phytoalexin; Kinetics; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Medicago - enzymology; Medicago - genetics; Medicago - metabolism; Medicago sativa; Medicago truncatula; Metabolism; Metabolism. Physicochemical requirements; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; Multigene Family - genetics; Phylogeny; Plant physiology and development; Plant Proteins - genetics; Plant Proteins - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Sequence Homology, Amino Acid; Substrate Specificity; elicitation; Medicago truncatula; Heterologous system; Hydroxylation; Cytochrome P450; Genomics; Biological model; Biosynthesis; Gene expression; Metabolism; Isoflavone derivatives; Structure function relationship; insect herbivory; Leguminosae; Phytoalexin; isoflavonoid phytoalexin; Dicotyledones; Angiospermae; Spermatophyta; functional expression
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313X.2003.01893.x

Summary 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.