Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorghum bicolor

Glucosinolates are natural products in cruciferous plants, including Arabidopsis thaliana. CYP79A1 is the cytochrome P450 catalysing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin in sorghum. Both glucosinolates and cyanogenic glucosides have oximes as intermediates. Expression of CYP79A1 in A. thaliana results in the production of high levels of the tyrosine-derived glucosinolate p-hydroxybenzylglucosinolate, which is not a natural constituent of A. thaliana. This provides further evidence that the enzymes have low substrate specificity with respect to the side chain. The ability of the cyanogenic CYP79A1 to integrate itself into the glucosinolate pathway has important implications for an evolutionary relationship between cyanogenic glucosides and glucosinolates, and for the possibility of genetic engineering of novel glucosinolates.     

Diversification of an ancient theme: hydroxynitrile glucosides

Many plants produce cyanogenic glucosides as part of their chemical defense. They are alpha-hydroxynitrile glucosides, which release toxic hydrogen cyanide (HCN) upon cleavage by endogenous plant beta-glucosidases. In addition to cyanogenic glucosides, several plant species produce beta- and gamma-hydroxynitrile glucosides. These do not release HCN upon hydrolysis by beta-glucosidases and little is known about their biosynthesis and biological significance. We have isolated three beta-hydroxynitrile glucosides, namely (2Z)-2-(beta-D-glucopyranosyloxy)but-2-enenitrile and (2R,3R)- and (2R,3S)-2-methyl-3-(beta-D-glucopyranosyloxy)butanenitrile, from leaves of Ribesuva-crispa. These compounds have not been identified previously. We show that in several species of the genera Ribes, Rhodiola and Lotus, these beta-hydroxynitrile glucosides co-occur with the L-isoleucine-derived hydroxynitrile glucosides, lotaustralin (alpha-hydroxynitrile glucoside), rhodiocyanosides A (gamma-hydroxynitrile glucoside) and D (beta-hydroxynitrile glucoside) and in some cases with sarmentosin (a hydroxylated rhodiocyanoside A). Radiolabelling experiments demonstrated that the hydroxynitrile glucosides in R. uva-crispa and Hordeum vulgare are derived from L-isoleucine and L-leucine, respectively. Metabolite profiling of the natural variation in the content of cyanogenic glucosides and beta- and gamma-hydroxynitrile glucosides in wild accessions of Lotus japonicus in combination with genetic crosses and analyses of the metabolite profile of the F2 population provided evidence that a single recessive genetic trait is most likely responsible for the presence or absence of beta- and gamma-hydroxynitrile glucosides in L. japonicus. Our findings strongly support the notion that the beta- and gamma-hydroxynitrile glucosides are produced by diversification of the cyanogenic glucoside biosynthetic pathway at the level of the nitrile intermediate.     

Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway

Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific β-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 (cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava (Manihot esculenta) and sorghum (Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario.     

Aldoxime-Forming Microsomal Enzyme Systems Involved in the Biosynthesis of Glucosinolates in Oilseed Rape (Brassica napus) Leaves

Glucosinolates and cyanogenic glucosides are synthesized from amino acids via similar intermediates, N-hydroxyamino acids and aldoximes. Microsomal preparations from young green leaves of oilseed rape catalyze the NADPH-dependent metabolism of homo-phenylalanine and dihomomethionine to the respective aldoximes, precursors of 2-phenylethyl and 3-butenyl glucosinolates. Cytochrome P-450-type enzymes are not involved (in contrast to cyanogenic glucoside biosynthesis), because neither activity was affected by carbon monoxide or other cytochrome P-450 inhibitors. Copper ions and diethyl pyrocarbonate were potent inhibitors of the enzymes, and treatment of microsomes with detergents abolished the overall activity. Two distinct enzyme systems with similar properties appear to be involved, each specific for a particular substrate. One utilizes dihomomethionine and is not active with homophenylalanine or any other amino acid tested, and the other is specific for homophenylalanine. From the characteristics of these enzymes, it seems that these early steps in glucosinolate biosynthesis may be catalyzed by flavin-containing monooxygenases comparable to those found in mammalian tissues and elsewhere. The pathways for the biosynthesis of glucosinolates and cyanogenic glucosides have apparently evolved independently, despite the similar chemical conversions involved.     

Hydroxynitrile glucosides

β- and γ-Hydroxynitrile glucosides are structurally related to cyanogenic glucosides (α-hydroxynitrile glucosides) but do not give rise to hydrogen cyanide release upon hydrolysis. Structural similarities and frequent co-occurrence suggest that the biosynthetic pathways for these compounds share common features. Based on available literature data we propose that oximes produced by CYP79 orthologs are common intermediates and that their conversion into β- and γ-hydroxynitrile glucosides is mediated by evolutionary diversified multifunctional orthologs to CYP71E1. We designate these as CYP71_βγ and CYP71_αβγ; in combination with the classical CYP71_α (CYP71E1 and orthologs) these are able to hydroxylate any of the carbon atoms present in the amino acid and oxime derived nitriles. Subsequent dehydration reactions and hydroxylations and a final glycosylation step afford the unsaturated β- and γ-hydroxynitrile glucosides. This scheme would explain the distribution patterns of α-, β- and γ-hydroxynitrile glucosides found in plants. The possible biological functions of these hydroxynitriles are discussed.     

Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid–derived cyanogenic glucosides (α-hydroxynitrile glucosides) by specific β-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores. We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled. Catabolic mutants could be placed in two complementation groups, one of which, cyd2, encoded the β-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related β-glucosidase, BGD4, were identified. This indicated that BGD4 plays no role in cyanogenesis in L. japonicus in vivo. Biochemical analysis confirmed that BGD4 cannot hydrolyze linamarin or lotaustralin and in L. japonicus is specific for breakdown of related hydroxynitrile glucosides, such as rhodiocyanoside A. By contrast, BGD2 can hydrolyze both cyanogenic glucosides and rhodiocyanosides. Our genetic analysis demonstrated specificity in the catabolic pathways for hydroxynitrile glucosides and implied specificity in their biosynthetic pathways as well. In addition, it has provided important tools for elucidating and potentially modifying cyanogenesis pathways in plants.     

Isolation of a Microsomal Enzyme System Involved in Glucosinolate Biosynthesis from Seedlings of Tropaeolum majus L

An in vitro system that converts phenylalanine to phenylacetaldoxime in the biosynthesis of the glucosinolate glucotropaeolin has been established in seedlings of Tropaeolum majus L. exposed to the combined treatment of jasmonic acid, ethanol, and light. The treatment resulted in a 9-fold induction, compared with untreated, dark-grown seedlings, of de novo biosynthesis measured as incorporation of radioactively labeled phenylalanine into glucotropaeolin. Formation of the inhibitory degradation product benzylisothiocyanate during tissue homogenization was prevented by inactivation of the thioglucosidase myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. This allowed the isolation of a biosynthetically active microsomal preparation from the induced T. majus plant material. The enzyme, which catalyzes the conversion of phenylalanine to the corresponding oxime, was sensitive to cytochrome P450 inhibitors, indicating the involvement of a cytochrome P450 in the biosynthetic pathway. It has previously been shown that the oxime-producing enzyme in the biosynthesis of p-hydroxybenzylglucosinolate in Sinapis alba L. is dependent on cytochrome P450, whereas the oxime-producing enzymes in Brassica species have been suggested to be flavin monooxygenases or peroxidase-type enzymes. The result with T. majus provides additional experimental documentation for a similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glucosides.     

CYP83b1 is the oxime-metabolizing enzyme in the glucosinolate pathway in Arabidopsis

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.     

Cyanogenic glycosides: a case study for evolution and application of cytochromes P450

Cyanogenic glycosides are ancient biomolecules found in more than 2,650 higher plant species as well as in a few arthropod species. Cyanogenic glycosides are amino acid-derived β-glycosides of α-hydroxynitriles. In analogy to cyanogenic plants, cyanogenic arthropods may use cyanogenic glycosides as defence compounds. Many of these arthropod species have been shown to de novo synthesize cyanogenic glycosides by biochemical pathways that involve identical intermediates to those known from plants, while the ability to sequester cyanogenic glycosides appears to be restricted to Lepidopteran species. In plants, two atypical multifunctional cytochromes P450 and a soluble family 1 glycosyltransferase form a metabolon to facilitate channelling of the otherwise toxic and reactive intermediates to the end product in the pathway, the cyanogenic glycoside. The glucosinolate pathway present in Brassicales and the pathway for cyanoalk(en)yl glucoside synthesis such as rhodiocyanosides A and D in Lotus japonicus exemplify how cytochromes P450 in the course of evolution may be recruited for novel pathways. The use of metabolic engineering using cytochromes P450 involved in biosynthesis of cyanogenic glycosides allows for the generation of acyanogenic cassava plants or cyanogenic Arabidopsis thaliana plants as well as L. japonicus and A. thaliana plants with altered cyanogenic, cyanoalkenyl or glucosinolate profiles.     

Leucine-derived cyano glucosides in barley

Barley (Hordeum vulgare) seedlings contain five cyano glucosides derived from the amino acid L-leucine (Leu). The chemical structure and the relative abundance of the cyano glucosides were investigated by liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses using spring barley cultivars with high, medium, and low cyanide potential. The barley cultivars showed a 10-fold difference in their cyano glucoside content, but the relative content of the individual cyano glucosides remained constant. Epiheterodendrin, the only cyanogenic glucoside present, comprised 12% to 18% of the total content of cyano glucosides. It is proposed that the aglycones of all five cyano glucosides are formed by the initial action of a cytochrome P450 enzyme of the CYP79 family converting L-Leu into Z-3-methylbutanal oxime and subsequent action of a less specific CYP71E enzyme converting the oxime into 3-methylbutyro nitrile and mediating subsequent hydroxylations at the alpha-, as well as beta- and gamma-, carbon atoms. Presence of cyano glucosides in the barley seedlings was restricted to leaf tissue, with 99% confined to the epidermis cell layers of the leaf blade. Microsomal preparations from epidermal cells were not able to convert L-[(14)C]Leu into the biosynthetic intermediate, Z-3-methylbutanal-oxime. This was only achieved using microsomal preparations from other cell types in the basal leaf segment, demonstrating translocation of the cyano glucosides to the epidermal cell layers after biosynthesis. A beta-glucosidase able to degrade epiheterodendrin was detected exclusively in yet a third compartment, the endosperm of the germinating seed. Therefore, in barley, a putative function of cyano glucosides in plant defense is not linked to cyanide release.     

Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin

A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450 – (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG – was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained.Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH–cytochrome P450–reductase in L--dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction.CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450–reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.     

Cytochrome P450 CYP79B2 from Arabidopsis catalyzes the conversion of tryptophan to indole-3-acetaldoxime, a precursor of indole glucosinolates and indole-3-acetic acid

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 microm 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-Müller, 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.     

Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. Catalyzes the conversion of L-phenylalanine to phenylacetaldoxime in the biosynthesis of benzylglucosinolate

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.     

The evolutionary appearance of non‐cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous β–glucosidases resulting from a crucial amino acid substitution

Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic α–hydroxynitrile glucosides lotaustralin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific β–glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non‐cyanogenic γ‐ and β–hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only β–glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site‐directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non‐flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic β–glucosidase, resembling BGD2, and required no more than a single amino acid substitution.     

CYANOGENIC GLYCOSIDES OF CARICA PAPAYA AND ITS PHYLOGENETIC POSITION WITH RESPECT TO THE VIOLALES AND CAPPARALES

Carica papaya L. (Caricaceae) was found to contain the cyclopentene-ring containing cyanogenic glucoside tetraphyllin B as well as the aromatic cyanogenic glucoside prunasin. This is the first report of the isolation of cyanogenic glucosides from a species known to produce glucosinolates. The presence of both classes of compounds suggests that Carica papaya may be intermediate between the Capparales and the Violales.     

Biosynthesis of the leucine derived α‐, β‐ and γ‐hydroxynitrile glucosides in barley (Hordeum vulgare L.)

Barley (Hordeum vulgare L.) produces five leucine‐derived hydroxynitrile glucosides (HNGs), of which only epiheterodendrin is a cyanogenic glucoside. The four non‐cyanogenic HNGs are the β‐HNG epidermin and the γ‐HNGs osmaronin, dihydroosmaronin and sutherlandin. By analyzing 247 spring barley lines including landraces and old and modern cultivars, we demonstrated that the HNG level varies notably between lines whereas the overall ratio between the compounds is constant. Based on sequence similarity to the sorghum (Sorghum bicolor) genes involved in dhurrin biosynthesis, we identified a gene cluster on barley chromosome 1 putatively harboring genes that encode enzymes in HNG biosynthesis. Candidate genes were functionally characterized by transient expression in Nicotiana benthamiana. Five multifunctional P450s, including two CYP79 family enzymes and three CYP71 family enzymes, and a single UDP‐glucosyltransferase were found to catalyze the reactions required for biosynthesis of all five barley HNGs. Two of the CYP71 enzymes needed to be co‐expressed for the last hydroxylation step in sutherlandin synthesis to proceed. This observation, together with the constant ratio between the different HNGs, suggested that HNG synthesis in barley is organized within a single multi‐enzyme complex.     

Cyanogenic glucosides in grapevine: polymorphism, identification and developmental patterns

Twelve grapevine (Vitis vinifera L.) cultivars were surveyed for 'cyanide potential' (i.e. the total cyanide measured in beta-glucosidase-treated crude, boiled tissue extract) in mature leaves. Two related cultivars (Carignan and Ruby Cabernet) had mean cyanide potential (equivalent to 110 mgHCNkg-1fr.wt) ca. 25-fold greater than that of the other 10 cultivars, and so the trait is polymorphic in the species. In boiled leaf extracts of Carignan and Ruby Cabernet, free cyanide constituted a negligible fraction of the total cyanide potential because beta-glucosidase treatment was required to liberate the major cyanide fraction - which is therefore bound in glucosylated cyanogenic compound(s) (or cyanogenic glucosides). In addition, cyanide was liberated from ground leaf tissue of Ruby Cabernet but not Sultana (a cultivar with low cyanide potential). Hence, the high cyanide potential in Ruby Cabernet leaves is coupled with endogenous beta-glucosidase(s) activity and this cultivar may be considered 'cyanogenic'. A method was developed to detect and identify cyanogenic glucosides using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). Two putative cyanogenic glucosides were found in extracts from leaves of Carignan and Ruby Cabernet and were identified as the epimers prunasin and sambunigrin. Cyanide potential measured at three times over the growing season in young and mature leaves, petioles, tendrils, flowers, berries, seeds and roots of Ruby Cabernet was substantially higher in the leaves compared with all other tissues. This characterisation of cyanogenic glucoside accumulation in grapevine provides a basis for gauging the involvement of the trait in interactions of the species with its pests and pathogens.     

Isolation and reconstitution of cytochrome P450ox and in vitro reconstitution of the entire biosynthetic pathway of the cyanogenic glucoside dhurrin from sorghum.

A cytochrome P450, designated P450ox, that catalyzes the conversion of (Z)-p-hydroxyphenylacetaldoxime (oxime) to p-hydroxymandelonitrile in the biosynthesis of the cyanogenic glucoside beta-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile (dhurrin), has been isolated from microsomes prepared from etiolated seedlings of sorghum (Sorghum bicolor L. Moench). P450ox was solubilized using nonionic detergents, and isolated by ion-exchange chromatography, Triton X-114 phase partitioning, and dye-column chromatography. P450ox has an apparent molecular mass of 55 kD, its N-terminal amino acid sequence is -ATTATPQLLGGSVP, and it contains the internal sequence MDRLVADLDRAAA. Reconstitution of P450ox with NADPH-P450 oxidoreductase in micelles of L-alpha-dilauroyl phosphatidylcholine identified P450ox as a multifunctional P450 catalyzing dehydration of (Z)-oxime to p-hydroxyphenylaceto-nitrile (nitrile) and C-hydroxylation of p-hydroxyphenylacetonitrile to nitrile. P450ox is extremely labile compared with the P450s previously isolated from sorghum. When P450ox is reconstituted in the presence of a soluble uridine diphosphate glucose glucosyltransferase, oxime is converted to dhurrin. In vitro reconstitution of the entire dhurrin biosynthetic pathway from tyrosine was accomplished by the insertion of CYP79 (tyrosine N-hydroxylase), P450ox, and NADPH-P450 oxidoreductase in lipid micelles in the presence of uridine diphosphate glucose glucosyltransferase. The catalysis of the conversion of Tyr into nitrile by two multifunctional P450s explains why all intermediates in this pathway except (Z)-oxime are channeled.     

Forward Genetics by Genome Sequencing Reveals That Rapid Cyanide Release Deters Insect Herbivory of Sorghum bicolor

Whole genome sequencing has allowed rapid progress in the application of forward genetics in model species. In this study, we demonstrated an application of next-generation sequencing for forward genetics in a complex crop genome. We sequenced an ethyl methanesulfonate-induced mutant of Sorghum bicolor defective in hydrogen cyanide release and identified the causal mutation. A workflow identified the causal polymorphism relative to the reference BTx623 genome by integrating data from single nucleotide polymorphism identification, prior information about candidate gene(s) implicated in cyanogenesis, mutation spectra, and polymorphisms likely to affect phenotypic changes. A point mutation resulting in a premature stop codon in the coding sequence of dhurrinase2, which encodes a protein involved in the dhurrin catabolic pathway, was responsible for the acyanogenic phenotype. Cyanogenic glucosides are not cyanogenic compounds but their cyanohydrins derivatives do release cyanide. The mutant accumulated the glucoside, dhurrin, but failed to efficiently release cyanide upon tissue disruption. Thus, we tested the effects of cyanide release on insect herbivory in a genetic background in which accumulation of cyanogenic glucoside is unchanged. Insect preference choice experiments and herbivory measurements demonstrate a deterrent effect of cyanide release capacity, even in the presence of wild-type levels of cyanogenic glucoside accumulation. Our gene cloning method substantiates the value of (1) a sequenced genome, (2) a strongly penetrant and easily measurable phenotype, and (3) a workflow to pinpoint a causal mutation in crop genomes and accelerate in the discovery of gene function in the postgenomic era.