title: CYP83A1 and CYP83B1, two nonredundant cytochrome P450 enzymes metabolizing oximes in the biosynthesis of glucosinolates in Arabidopsis.
abstract: In the glucosinolate pathway, the postoxime enzymes have been proposed to have low specificity for the side chain and high specificity for the functional group. Here, we provide biochemical evidence for the functional role of the two cytochromes P450, CYP83A1 and CYP83B1, from Arabidopsis in oxime metabolism in the biosynthesis of glucosinolates. In a detailed analysis of the substrate specificities of the recombinant enzymes heterologously expressed in yeast (Saccharomyces cerevisiae), we show that aliphatic oximes derived from chain-elongated homologs of methionine are efficiently metabolized by CYP83A1, whereas CYP83B1 metabolizes these substrates with very low efficiency. Aromatic oximes derived from phenylalanine, tryptophan, and tyrosine are metabolized by both enzymes, although CYP83B1 has higher affinity for these substrates than CYP83A1, particularly in the case of indole-3-acetaldoxime, where there is a 50-fold difference in K(m) value. The data show that CYP83A1 and CYP83B1 are nonredundant enzymes under physiologically normal conditions in the plant. The ability of CYP83A1 to metabolize aromatic oximes, albeit at small levels, explains the presence of indole glucosinolates at various levels in different developmental stages of the CYP83B1 knockout mutant, rnt1-1. Plants overexpressing CYP83B1 contain elevated levels of aliphatic glucosinolates derived from methionine homologs, whereas the level of indole glucosinolates is almost constant in the overexpressing lines. Together with the previous characterization of the members of the CYP79 family involved in oxime production, this work provides a framework for metabolic engineering of glucosinolates and for further dissection of the glucosinolate pathway.
authors.affiliation: Plant Biochemistry Laboratory, Department of Chemistry, Center for Molecular Plant Physiology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Naur
authors.forename: Bent Larsen
authors.initials: BL
authors.lastname: Petersen
authors.forename: Michael Dalgaard
authors.initials: MD
authors.lastname: Mikkelsen
authors.forename: Søren
authors.initials: S
authors.lastname: Bak
authors.forename: Hasse
authors.initials: H
authors.lastname: Rasmussen
authors.forename: Carl Erik
authors.initials: CE
authors.lastname: Olsen
authors.forename: Barbara Ann
authors.initials: BA
authors.lastname: Halkier
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005961:Glucosinolates / Q000096:biosynthesis*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010091:Oximes / Q000378:metabolism*; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D029681:Arabidopsis Proteins; D005961:Glucosinolates; D010091:Oximes; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C094565:CYP83A1 protein, Arabidopsis; C431937:CYP83B1 protein, Arabidopsis