First published online August 7, 2003; 10.1104/pp.102.019240
Plant Physiology 133:63-72 (2003)
© 2003 American Society of Plant Biologists
BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES
CYP83A1 and CYP83B1, Two Nonredundant Cytochrome P450 Enzymes Metabolizing Oximes in the Biosynthesis of Glucosinolates in Arabidopsis1
Peter Naur2,
Bent Larsen Petersen3,
Michael Dalgaard Mikkelsen,
Søren Bak,
Hasse Rasmussen,
Carl Erik Olsen and
Barbara Ann Halkier*
Plant Biochemistry Laboratory (P.N., B.L.P., M.D.M., S.B., B.A.H.) and Department of Chemistry (C.E.O.), Center for Molecular Plant Physiology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; and IACR-Rothamsted, Harpenden AL5 2JQ, United Kingdom (H.R.)
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 Km 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.
1 This work was supported by grants from the Danish National Research Foundation and the Director Ib Henriksens Foundation.
2 Present address: Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark.
3 Present address: Danish Institute of Agricultural Sciences, Biotechnology Group, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
* Corresponding author; e-mail bah{at}kvl.dk; fax 45-35283333.
Received December 17, 2002;
returned for revision January 29, 2003;
accepted May 12, 2003.
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