title: The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism.
abstract: The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants; however, no conditions suitable for the unambiguous assay of the enzyme are known. As a result, all attempts to purify the protein and clone its corresponding gene have failed. By screening for plants that accumulate reduced levels of soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. Using radiotracer-feeding experiments, we have determined that the ref8 mutant is unable to synthesize caffeic acid, suggesting that the mutant is defective in a gene required for the activity or expression of C3H. We have isolated the REF8 gene using positional cloning methods, and have verified that it encodes C3H by expression of the wild-type gene in yeast. Although many previous reports in the literature have suggested that C3H is a phenolase, the isolation of the REF8 gene demonstrates that the enzyme is actually a cytochrome P450-dependent monooxygenase. Although the enzyme accepts p-coumarate as a substrate, it also exhibits significant activity towards other p-hydroxylated substrates. These data may explain the previous difficulties in identifying C3H activity in plant extracts and they indicate that the currently accepted version of the lignin biosynthetic pathway is likely to be incorrect.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN 47907-1153, USA.
authors.forename: Rochus
authors.initials: R
authors.lastname: Franke
authors.forename: John M
authors.initials: JM
authors.lastname: Humphreys
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Hemm
authors.forename: Jeff W
authors.initials: JW
authors.lastname: Denault
authors.forename: Max O
authors.initials: MO
authors.lastname: Ruegger
authors.forename: Joanne C
authors.initials: JC
authors.lastname: Cusumano
authors.forename: Clint
authors.initials: C
authors.lastname: Chapple
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins; D002109:Caffeic Acids / Q000378:metabolism; D002734:Chlorophyll / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003373:Coumaric Acids / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005030:Ethylenes / Q000378:metabolism; D005453:Fluorescence; D005816:Genetic Complementation Test; D008031:Lignin / Q000096:biosynthesis*; D008293:Malates / Q000302:isolation & purification / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D014442:Monophenol Monooxygenase; D009154:Mutation; D010666:Phenylpropionates / Q000302:isolation & purification / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: C079164:2-O-sinapoylmalate; D029681:Arabidopsis Proteins; D002109:Caffeic Acids; D003373:Coumaric Acids; D005030:Ethylenes; D008293:Malates; D010666:Phenylpropionates; D002734:Chlorophyll; C073734:sinapinic acid; D008031:Lignin; D003577:Cytochrome P-450 Enzyme System; C036216:ethylene; D006899:Mixed Function Oxygenases; C438015:CYP98A3 protein, Arabidopsis; D014442:Monophenol Monooxygenase; C040048:caffeic acid