The presence of CYP79 homologues in glucosinolate-producing plants shows evolutionary conservation of the enzymes in the conversion of amino acid to aldoxime in the biosynthesis of cyanogenic glucosides and glucosinolates

A cDNA encoding CYP79B1 has been isolated from Sinapis alba. CYP79B1 from S. alba shows 54% sequence identity and 73% similarity to sorghum CYP79A1 and 95% sequence identity to the Arabidopsis T42902, assigned CYP79B2. The high identity and similarity to sorghum CYP79A1, which catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin, suggests that CYP79B1 similarly catalyses the conversion of amino acid(s) to aldoxime(s) in the biosynthesis of glucosinolates. Within the highly conserved ‘PERF’ and the heme-binding region of A-type cytochromes, the CYP79 family has unique substitutions that define the family-specific consensus sequences of FXP(E/D)RH and SFSTG(K/R)RGC(A/I)A, respectively. Sequence analysis of PCR products generated with CYP79B subfamily-specific primers identified CYP79B homologues in Tropaeolum majus, Carica papaya, Arabidopsis, Brassica napus and S. alba. The five glucosinolate-producing plants identified a CYP79B amino acid consensus sequence KPERHLNECSEVTLTENDLRFISFSTGKRGC. The unique substitutions in the ‘PERF’ and the heme-binding domain and the high sequence identity and similarity of CYP79B1, CYP79B2 and CYP79A1, together with the isolation of CYP79B homologues in the distantly related Tropaeolaceae, Caricaceae and Brassicaceae within the Capparales order, show that the initial part of the biosynthetic pathway of glucosinolates and cyanogenic glucosides is catalysed by evolutionarily conserved cytochromes P450. This confirms that the appearance of glucosinolates in Capparales is based on a cyanogen ‘predisposition’. Identification of CYP79 homologues in glucosinolate-producing plants provides an important tool for tissue-specific regulation of the level of glucosinolates to improve nutritional value and pest resistance.

[1]  B. Halkier,et al.  Isolation of the heme-thiolate enzyme cytochrome P-450TYR, which catalyzes the committed step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[2]  B. Halkier,et al.  Purification and characterization of recombinant cytochrome P450TYR expressed at high levels in Escherichia coli. , 1995, Archives of biochemistry and biophysics.

[3]  M. Schuler.  Plant Cytochrome P450 Monooxygenases , 1996 .

[4]  D. Reed,et al.  The Identification of Desulfoglucosinolates Using Thermospray Liquid Chromatography/Mass Spectrometry , 1988 .

[5]  B. Halkier,et al.  Cloning and expression in Escherichia coli of the obtusifoliol 14 alpha-demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14 alpha-demethylases (CYP51) from fungi and mammals. , 1997, The Plant journal : for cell and molecular biology.

[6]  J. Ludwig-Müller,et al.  A plasma membrane-bound enzyme oxidizes L-tryptophan to indole-3-acetaldoxime. , 1988 .

[7]  R. Bennett,et al.  Biosynthesis of benzylglucosinolate, cyanogenic glucosides and phenylpropanoids in Carica papaya , 1997 .

[8]  J. Rodman,et al.  Molecules, morphology, and Dahlgren's expanded order Capparales , 1996 .

[9]  B. Halkier,et al.  Isolation of a Microsomal Enzyme System Involved in Glucosinolate Biosynthesis from Seedlings of Tropaeolum majus L , 1996, Plant physiology.

[10]  B. Halkier,et al.  The primary sequence of cytochrome P450tyr, the multifunctional N-hydroxylase catalyzing the conversion of L-tyrosine to p-hydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench. , 1995, Archives of biochemistry and biophysics.

[11]  R. Bennett,et al.  Aldoxime-Forming Microsomal Enzyme Systems Involved in the Biosynthesis of Glucosinolates in Oilseed Rape (Brassica napus) Leaves , 1993, Plant physiology.

[12]  R. Bennett,et al.  Involvement of Cytochrome P450 in Glucosinolate Biosynthesis in White Mustard (A Biochemical Anomaly) , 1997, Plant physiology.

[13]  Catalytic reactivities and struc-ture/function relationships of cytochrome P490 enzymes , 1996 .

[14]  H. Barnes,et al.  Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[15]  R. Bennett,et al.  Distribution and activity of microsomal NADPH-dependent monooxygenases and amino acid decarboxylases in cruciferous and non-cruciferous plants, and their relationship to foliar glucosinolate content , 1996 .

[16]  B. Halkier,et al.  Characterization of Cytochrome P450TYR, A Multifunctional Haem-Thiolate AZ-Hydroxylase Involved in the Biosynthesis of the Cyanogenic Glucoside Dhurrin , 1995, Drug metabolism and drug interactions.

[17]  V. Šedivec,et al.  [Sulfur compounds in the air of viscose rayon plants]. , 1953, Pracovni lekarstvi.

[18]  B. Halkier,et al.  Cytochrome P-450TYR Is a Multifunctional Heme-Thiolate Enzyme Catalyzing the Conversion of L-Tyrosine to p-Hydroxyphenylacetaldehyde Oxime in the Biosynthesis of the Cyanogenic Glucoside Dhurrin in Sorghum bicolor (L.) Moench (*) , 1995, The Journal of Biological Chemistry.

[19]  B. Halkier,et al.  Involvement of cytochrome P450 in oxime production in glucosinolate biosynthesis as demonstrated by an in vitro microsomal enzyme system isolated from jasmonic acid-induced seedlings of Sinapis alba L. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[20]  B. Halkier,et al.  Isolation and Reconstitution of Cytochrome P450ox and in Vitro Reconstitution of the Entire Biosynthetic Pathway of the Cyanogenic Glucoside Dhurrin from Sorghum , 1997, Plant physiology.

[21]  B. Halkier,et al.  The biosynthesis of glucosinolates , 1997 .

[22]  J Deisenhofer,et al.  Structure and function of cytochromes P450: a comparative analysis of three crystal structures. , 1995, Structure.

[23]  B. Halkier,et al.  The biosynthesis of cyanogenic glucosides in seedlings of cassava (Manihot esculenta Crantz). , 1992, Archives of biochemistry and biophysics.

[24]  H. Barnes.  Maximizing expression of eukaryotic cytochrome P450s in Escherichia coli. , 1996, Methods in enzymology.

[25]  R. Bennett,et al.  Glucosinolate Biosynthesis (Further Characterization of the Aldoxime-Forming Microsomal Monooxygenases in Oilseed Rape Leaves) , 1995, Plant physiology.

[26]  D. Nelson,et al.  Diversity and Evolution of Plant P450 and P450-Reductases , 1995, Drug metabolism and drug interactions.

[27]  H. Sørensen.  Glucosinolates: Structure-Properties-Function , 1990 .