Glucosinolates are natural plant products known as flavor compounds, cancer-preventing agents, and biopesticides. We report cloning and characterization of the cytochrome P450 CYP79B2 from Arabidopsis. Heterologous expression of CYP79B2 in Escherichia coli shows that CYP79B2 catalyzes the conversion of tryptophan to indole-3-acetaldoxime. Recombinant CYP79B2 has a K m of 21 M and a V max of 7.78 nmol/h/ml culture. Inhibitor studies show that CYP79B2 is different from a previously described enzyme activity that converts tryptophan to indole-3-acetaldoxime (Ludwig-Mü ller, J., and Hilgenberg, W. (1990) Phytochemistry, 29, 1397-1400). CYP79B2 is wound-inducible and expressed in leaves, stem, flowers, and roots, with the highest expression in roots. Arabidopsis overexpressing CYP79B2 has increased levels of indole glucosinolates, which strongly indicates that CYP79B2 is involved in indole glucosinolate biosynthesis. Our data show that oxime production by CYP79s is not restricted to those amino acids that are precursors for cyanogenic glucosides. Our data are consistent with the hypothesis that indole glucosinolates have evolved from cyanogenesis. Indole-3-acetaldoxime is a precursor of the plant hormone indole-3-acetic acid, which suggests that CYP79B2 might function in biosynthesis of indole-3-acetic acid. Identification of CYP79B2 provides an important tool for modification of the indole glucosinolate content to improve nutritional value and pest resistance.Glucosinolates are natural plant products characterized by having a thioglucose moiety, a sulfonated oxime, and a side chain derived from aliphatic, aromatic, and indole amino acids. Glucosinolates are found in the order Capparales, where they co-occur with the endogenous thioglucosidase myrosinase (for review see Ref. 1). Generally, the glucosinolate-myrosinase system is believed to play an important role in plant defense. In human affairs, glucosinolates are important as flavor compounds, cancer-preventive agents, and biopesticides, for example. There is a strong interest in controlling the level of glucosinolates to improve flavor and nutritional qualities of food crops and to study the physiological role of glucosinolates in plants, e.g. in plant-pest interaction.Glucosinolates are related to cyanogenic glucosides as both groups of natural plant products are derived from amino acids and have oximes as intermediates. Cytochromes P450 of the CYP79 family have been shown to catalyze the conversion of both aliphatic and aromatic amino acids to their corresponding oximes in the biosynthesis of cyanogenic glucosides (2-4). Recently, we have shown that the aromatic amino acid phenylalanine is converted to its oxime by CYP79A2 from Arabidopsis (5). This is in accordance with biochemical data from microsomal enzyme systems isolated from the glucosinolate-producing Sinapis alba, Tropaeolum majus, and Carica papaya (6 -10).The nature of the enzymes catalyzing oxime production in the biosynthesis of glucosinolates has been subject of discussions as independent studies ...

The glucosinolate composition and content in various tissues of Arabidopsis thaliana (L.) Heynh. ecotype Columbia during development from seeds to bolting plants were determined in detail by high-performance liquid chromatography. Comparison of the glucosinolate profiles of leaves, roots and stems from mature plants with those of green siliques and mature seeds indicated that a majority of the seed glucosinolates were synthesized de novo in the silique. A comparison of the glucosinolate profile of mature seeds with that of cotyledons indicated that a major part of seed glucosinolates was retained in the cotyledons. Turnover of glucosinolates was studied by germination of seeds containing radiolabelled p-hydroxybenzylglucosinolate (p-OHBG). Approximately 70% of the content of [14C]p-OHBG in the seeds was detected in seedlings at the cotyledon stage and [14C]p-OHBG was barely detectable in young plants with rosettes of six to eight leaves. The turn-over of p-OHBG was found to coincide with the expression of the glucosinolate-degrading enzyme myrosinase, which was detectable at very low levels in seedlings at the cotyledon stage, but which dramatically increased in leaves from plants at later developmental stages. This indicates that there is a continuous turnover of glucosinolates during development and not only upon tissue disruption.

SummaryCytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono-to hexahomomethionine, resulting in both short-and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta-and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.

Glucosinolates are natural plant products that have received rising attention due to their role in interactions between pests and crop plants and as chemical protectors against cancer. Glucosinolates are derived from amino acids and have aldoximes as intermediates. We report that cytochrome P450 CYP79F1 catalyzes aldoxime formation in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 functionally expressed in Escherichia coli, we show that both dihomomethionine and trihomomethionine are metabolized by CYP79F1 resulting in the formation of 5-methylthiopentanaldoxime and 6-methylthiohexanaldoxime, respectively. 5-methylthiopentanaldoxime is the precursor of the major glucosinolates in leaves of A. thaliana, i.e. 4-methylthiobutylglucosinolate and 4-methylsulfinylbutylglucosinolate, and a variety of other glucosinolates in Brassica sp. Transgenic A. thaliana with cosuppression of CYP79F1 have a reduced content of aliphatic glucosinolates and a highly increased level of dihomomethionine and trihomomethionine. The transgenic plants have a morphological phenotype showing loss of apical dominance and formation of multiple axillary shoots. Our data provide the first evidence that a cytochrome P450 catalyzes the Nhydroxylation of chain-elongated methionine homologues to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates.

and the ‡ ‡IACR-Rothamsted, Harpenden, AL5 2JQ, United Kingdom CYP83B1 from Arabidopsis thaliana has been identified as the oxime-metabolizing enzyme in the biosynthetic pathway of glucosinolates. Biosynthetically active microsomes isolated from Sinapis alba converted p-hydroxyphenylacetaldoxime and cysteine into S-alkylated p-hydroxyphenylacetothiohydroximate, S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine, the next proposed intermediate in the glucosinolate pathway. The production was shown to be dependent on a cytochrome P450 monooxygenase. We searched the genome of A. thaliana for homologues of CYP71E1 (P450ox), the only known oxime-metabolizing enzyme in the biosynthetic pathway of the evolutionarily related cyanogenic glucosides. By a combined use of bioinformatics, published expression data, and knock-out phenotypes, we identified the cytochrome P450 CYP83B1 as the oxime-metabolizing enzyme in the glucosinolate pathway as evidenced by characterization of the recombinant protein expressed in Escherichia coli. The data are consistent with the hypothesis that the oxime-metabolizing enzyme in the cyanogenic pathway (P450ox) was mutated into a "P450mox" that converted oximes into toxic compounds that the plant detoxified into glucosinolates.Glucosinolates are naturally occurring amino acid-derived S-glucosides of thiohydroximate-O-sulfonates. They co-occur with endogenous thioglucosidases called myrosinases that upon tissue damage hydrolyze glucosinolates into a wide range of degradation products such as e.g. isothiocyanates, nitriles, and thiocyanates. Glucosinolates (or rather their degradation products) are involved in plant defense and constitute characteristic flavor compounds and cancer-preventive agents in Brassica vegetables.The biosynthetic pathway from precursor amino acid to the core glucosinolate structure has been well studied, and many of the intermediates are known, including oximes, thiohydroximic acids, and desulfoglucosinolates (1, 2). Recently, it has been shown that cytochromes P450 belonging to the CYP79 family catalyze the conversion of amino acids to oximes (3-7). Little is known about the formation of thiohydroximic acids from oximes. The remaining part of the pathway for the core structure involves a UDP-glucose:thiohydroximic acid glucosyltransferase and a sulfotransferase (for review, see Ref.2).It has been proposed that aci-nitro compounds are intermediates in the conversion of oximes to thiohydroximic acids (8). This was supported by isolation of 1-nitro-2-phenylethane from Tropaeolum majus shoots and by in vivo conversion of phenylacetaldoxime into 1-nitro-2-phenylethane and of 1-nitro-2-[1,2-14 C]phenylethane into benzylglucosinolate (9). The aci-nitro is proposed to be conjugated with a sulfur donor to produce an S-alkyl thiohydroximate, possibly by a glutathione S-transferase (2). Biochemical studies indicate that the S-alkyl thiohydroximate is subsequently hydrolyzed to the thiohydroximic acid by a C-S lyase (10).Glucosinolates are related to cyanogenic glucosides because both...