Cytochrome P450 CYP79F1 from Arabidopsis Catalyzes the Conversion of Dihomomethionine and Trihomomethionine to the Corresponding Aldoximes in the Biosynthesis of Aliphatic Glucosinolates

Abstract

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.

Variations in CYP74B2 (Hydroperoxide Lyase) Gene Expression Differentially Affect Hexenal Signaling in the Columbia and Landsberg erecta Ecotypes of Arabidopsis

“…The consistency of the low CYP83A1 transcript levels in the Col ecotype in both types of arrays as well as RT-PCR analysis confirm the prediction by Hemm et al (2003) that allelic variation at the CYP83A1 locus exists among Arabidopsis ecotypes. The contrasting higher transcript levels in the Col ecotype (2.3-fold) of CYP83B1, which mediates synthesis of indole glucosinolates Hansen et al, 2001), suggests that compensatory interplay exists between the CYP83A1 and CYP83B1 loci as previously seen in the studies of Hemm et al (2003) and Naur et al (2003). The differential expression of CYP83A1 in these ecotypes partially explains the different basal glucosinolate profiles in the Col and Ler ecotypes (Kliebenstein et al, 2001a) and the higher overall glucosinolate content of the Ler ecotype (Jander et al, 2001).…”

“…Together with the higher expression levels of CYP74B2 (HPL) transcripts and volatiles produced, these data suggest that the levels of JAs are also elevated in Ler leaves compared to Col leaves. Also interesting among the set more highly expressed in the Ler ecotype are several loci in aliphatic glucosinolate synthesis, including CYP79F1, which exists upstream of CYP83A1 (Hansen et al, 2001;Reintanz et al, 2001;Chen et al, 2003), CYP83A1 itself, and 2-oxoglutarate-dependent dioxygenase (AOP3), which exists downstream of CYP83A1 (Kliebenstein et al, 2001b); Trp synthetase (TSB2; Last et al, 1991) also potentially impacts aliphatic glucosinolate synthesis because it is postulated to feed substrates into the aliphatic glucosinolate pathway via CYP79B2 and CYP79B3 . Contrasting with these glucosinolate synthetic enzymes that are more highly expressed in the Ler ecotype, one locus more highly expressed in the Col ecotype codes for thioglycosyl hydrolase (TGG2, myrosinase), which degrades glucosinolates to release toxic derivatives (Xue et al, 1995).…”

Functional Analysis of the Tandem-Duplicated P450 Genes SPS/BUS/CYP79F1 and CYP79F2 in Glucosinolate Biosynthesis and Plant Development by Ds Transposition-Generated Double Mutants

“…These mutants exhibit massive proliferation of shoots, together with other developmental defects. The quantification of hormone levels in sps/cyp79F1 mutant plants indicates that both auxin and cytokinins are higher in the mutants, but the phenotypes of the sps/cyp79F1 plants are consistent with higher levels of cytokinins rather than auxins as the key factor responsible for the change in branching pattern (Tantikanjana et al, 2001).Despite the fact that sps/cyp79F1 plants resemble hormone mutants, biochemical studies have shown that SPS/CYP79F1 gene encodes an enzyme catalyzing metabolism of both short-chain and long-chain elongated Met-derivatives in the biosynthesis of aliphatic glucosinolates (Hansen et al, 2001;Chen et al, 2003). Glucosinolates are a group of secondary plant metabolites known to play a role in plant defense and are sources of flavor compounds, cancer-preventing agents, and bioherbicides.…”

“…Recently SPS/CYP79F1 and CYP79F2 genes have been shown to have distinct but overlapping metabolic functions. SPS/CYP79F1 metabolizes both short-chain and long-chain aliphatic Mets, whereas CYP79F2 exclusively metabolizes the long-chain elongated aliphatic Mets (Hansen et al, 2001;Chen et al, 2003). In order to reveal any possible overlapping physiological roles, as well as to confirm metabolic roles of the SPS/ CYP79F1 and CYP79F2 genes, we generated sps/ cyp79F1 and cyp79F2 double mutants by multiple Ds transpositions.…”

“…Despite the fact that sps/cyp79F1 plants resemble hormone mutants, biochemical studies have shown that SPS/CYP79F1 gene encodes an enzyme catalyzing metabolism of both short-chain and long-chain elongated Met-derivatives in the biosynthesis of aliphatic glucosinolates (Hansen et al, 2001;Chen et al, 2003). Glucosinolates are a group of secondary plant metabolites known to play a role in plant defense and are sources of flavor compounds, cancer-preventing agents, and bioherbicides.…”