Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. Catalyzes the Conversion of l-Phenylalanine to Phenylacetaldoxime in the Biosynthesis of Benzylglucosinolate

Ute Wittstock,Barbara Ann Halkier

doi:10.1074/jbc.275.19.14659

Abstract

Glucosinolates are natural plant products gaining increasing interest as cancer-preventing agents and crop protectants. Similar to cyanogenic glucosides, glucosinolates are derived from amino acids and have aldoximes as intermediates. We report cloning and characterization of cytochrome P450 CYP79A2 involved in aldoxime formation in the glucosinolate-producing Arabidopsis thaliana L. The CYP79A2 cDNA was cloned by polymerase chain reaction, and CYP79A2 was functionally expressed in Escherichia coli. Characterization of the recombinant protein shows that CYP79A2 is an N-hydroxylase converting L-phenylalanine into phenylacetaldoxime, the precursor of benzylglucosinolate. Transgenic A. thaliana constitutively expressing CYP79A2 accumulate high levels of benzylglucosinolate. CYP79A2 expressed in E. coli has a K(m) of 6.7 micromol liter(-1) for L-phenylalanine. Neither L-tyrosine, L-tryptophan, L-methionine, nor DL-homophenylalanine are metabolized by CYP79A2, indicating that the enzyme has a narrow substrate specificity. CYP79A2 is the first enzyme shown to catalyze the conversion of an amino acid to the aldoxime in the biosynthesis of glucosinolates. Our data provide the first conclusive evidence that evolutionarily conserved cytochromes P450 catalyze this step common for the biosynthetic pathways of glucosinolates and cyanogenic glucosides. This strongly indicates that the biosynthesis of glucosinolates has evolved based on a cyanogenic predisposition.

Highlights

Glucosinolates are natural plant products gaining increasing interest as cancer-preventing agents and crop protectants
We report cloning and characterization of CYP79A2, a cytochrome P450-dependent monooxygenase, which catalyzes the conversion of L-phenylalanine to phenylacetaldoxime in the biosynthesis of benzylglucosinolate in A. thaliana
Glucosinolates are related to cyanogenic glucosides as the biosynthesis of both classes of secondary plant products proceeds from amino acids via aldoximes

Summary

EXPERIMENTAL PROCEDURES

PCR Primers—The following PCR primers (Fig. 1) were designed from the genomic CYP79A2 sequence PCR reactions were set up in a total volume of 50 ␮l in Expand HF buffer with 1.5 mM MgCl2 (Roche Molecular Biochemicals) supplemented with 200 ␮M dNTPs, 50 pmol of each primer, and 5% (v/v) Me2SO. After incubation of the reactions at 97 °C for 3 min, 2.6 units of Expand High Fidelity PCR system (Roche Molecular Biochemicals) were added and 35 cycles of 90 s at 95 °C, 60 s at 65 °C, 120 s at 70 °C were run. Generation of Expression Constructs—Expression constructs were derived from a CYP79A2 cDNA, which had been obtained by fusion of the two CYP79A2 exons generated from genomic DNA of Arabidopsis thaliana L. The two exons were amplified by PCR with primers A2F2/ A2R3 and A2F3/A2R2, respectively, using 1.25 units of Pwo polymerase (Roche Molecular Biochemicals) and 4 ␮g of template DNA. PCR reactions were set up in a total volume of 50 ␮l in Pwo polymerase PCR

The abbreviations used are

RESULTS

DISCUSSION