title: Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. Catalyzes the conversion of L-phenylalanine to phenylacetaldoxime in the biosynthesis of benzylglucosinolate.
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.
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant Biology, and Center for Molecular Plant Physiology (PlaCe), The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: U
authors.initials: U
authors.lastname: Wittstock
authors.forename: B A
authors.initials: BA
authors.lastname: Halkier
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010091:Oximes / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013861:Thiocyanates / Q000378:metabolism*; D013863:Thioglucosides / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D010091:Oximes; D011994:Recombinant Proteins; D013861:Thiocyanates; D013863:Thioglucosides; C408882:benzylglucosinolic acid; D010649:Phenylalanine; C408881:phenylacetaldoxime; D003577:Cytochrome P-450 Enzyme System; C408883:CYP79A2 protein, Arabidopsis; D006899:Mixed Function Oxygenases