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.     

Determination of catalytic key amino acids and UDP sugar donor specificity of the cyanohydrin glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular modeling substantiated by site-specific mutagenesis and biochemical analyses

Plants produce a plethora of structurally diverse natural products. The final step in their biosynthesis is often a glycosylation step catalyzed by a family 1 glycosyltransferase (GT). In biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor, the UDP-glucosyltransferase UGT85B1 catalyzes the conversion of p-hydroxymandelonitrile into dhurrin. A structural model of UGT85B1 was built based on hydrophobic cluster analysis and the crystal structures of two bacterial GTs, GtfA and GtfB, which each showed approximately 15% overall amino acid sequence identity to UGT85B1. The model enabled predictions about amino acid residues important for catalysis and sugar donor specificity. p-Hydroxymandelonitrile and UDP-glucose (Glc) were predicted to be positioned within hydrogen-bonding distance to a glutamic acid residue in position 410 facilitating sugar transfer. The acceptor was packed within van der Waals distance to histidine H23. Serine S391 and arginine R201 form hydrogen bonds to the pyrophosphate part of UDP-Glc and hence stabilize binding of the sugar donor. Docking of UDP sugars predicted that UDP-Glc would serve as the sole donor sugar in UGT85B1. This was substantiated by biochemical analyses. The predictive power of the model was validated by site-directed mutagenesis of selected residues and using enzyme assays. The modeling approach has provided a tool to design GTs with new desired substrate specificities for use in biotechnological applications. The modeling identified a hypervariable loop (amino acid residues 156-188) that contained a hydrophobic patch. The involvement of this loop in mediating binding of UGT85B1 to cytochromes P450, CYP79A1, and CYP71E1 within a dhurrin metabolon is discussed.     

Homology modeling of the three membrane proteins of the dhurrin metabolon: catalytic sites, membrane surface association and protein-protein interactions

Formation of metabolons (macromolecular enzyme complexes) facilitates the channelling of substrates in biosynthetic pathways. Metabolon formation is a dynamic process in which transient structures mediated by weak protein-protein interactions are formed. In Sorghum, the cyanogenic glucoside dhurrin is derived from l-tyrosine in a pathway involving the two cytochromes P450 (CYPs) CYP79A1 and CYP71E1, a glucosyltransferase (UGT85B1), and the redox partner NADPH-dependent cytochrome P450 reductase (CPR). Experimental evidence suggests that the enzymes of this pathway form a metabolon. Homology modeling of the three membrane bound proteins was carried out using the Sybyl software and available relevant crystal structures. Residues involved in tight positioning of the substrates and intermediates in the active sites of CYP79A1 and CYP71E1 were identified. In both CYPs, hydrophobic surface domains close to the N-terminal trans-membrane anchor and between the F′ and G helices were identified as involved in membrane anchoring. The proximal surface of both CYPs showed positively charged patches complementary to a negatively charged bulge on CPR carrying the FMN domain. A patch of surface exposed, positively charged amino acid residues positioned on the opposite face of the membrane anchor was identified in CYP71E1 and might be involved in binding UGT85B1 via a hypervariable negatively charged loop in this protein.     

Substrate specificity of the cytochrome P450 enzymes CYP79A1 and CYP71E1 involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench

The two multifunctional cytochrome P450 enzymes, CYP79A1 and CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench have been characterized with respect to substrate specificity and cofactor requirements using reconstituted, recombinant enzymes and sorghum microsomes. CYP79A1 has a very high substrate specificity, tyrosine being the only substrate found. CYP71E1 has less stringent substrate requirements and metabolizes aromatic oximes efficiently, whereas aliphatic oximes are slowly metabolized. Neither CYP79A1 nor CYP71E1 catalyze the metabolism of a range of different herbicides. The reported resistance of sorghum to bentazon is therefore not linked to the presence of CYP79A1 or CYP71E1. NADPH is a much better cofactor than NADH although NADH does support the entire catalytic cycle of both P450 enzymes. Km and Vmax values for NADPH when supporting CYP71E1 activity are 0.013 mM and 111 nmol/mg protein/s. For NADH, the corresponding values are 0. 3 mM and 42 nmol/mg protein/s. CYP79A1 is a fairly stable enzyme. In contrast, CYP71E1 is labile and prone to rapid denaturation at room temperature. CYP71E1 is isolated in the low spin form. CYP71E1 catalyzes an unusual dehydration reaction of an oxime to the corresponding nitrile which subsequently is C-hydroxylated. The oxime forms a peculiar reverse Type I spectrum, whereas the nitrile forms a Type I spectrum. Several compounds which do not serve as substrates formed Type I substrate binding spectra with the two P450 enzymes.     

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.     

Cloning and expression of cytochrome P450 enzymes catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of cyanogenic glucosides in Triglochin maritima

Two cDNA clones encoding cytochrome P450 enzymes belonging to the CYP79 family have been isolated from Triglochin maritima. The two proteins show 94% sequence identity and have been designated CYP79E1 and CYP79E2. Heterologous expression of the native and the truncated forms of the two clones in Escherichia coli demonstrated that both encode multifunctional N-hydroxylases catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the two cyanogenic glucosides taxiphyllin and triglochinin in T. maritima. This renders CYP79E functionally identical to CYP79A1 from Sorghum bicolor, and unambiguously demonstrates that cyanogenic glucoside biosynthesis in T. maritima and S. bicolor is catalyzed by analogous enzyme systems with p-hydroxyphenylacetaldoxime as a free intermediate. This is in contrast to earlier reports stipulating p-hydroxyphenylacetonitrile as the only free intermediate in T. maritima. L-3,4-Dihydroxyphenyl[3-(14)C]Ala (DOPA) was not metabolized by CYP79E1, indicating that hydroxylation of the phenol ring at the meta position, as required for triglochinin formation, takes place at a later stage. In S. bicolor, CYP71E1 catalyzes the subsequent conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile. When CYP79E1 from T. maritima was reconstituted with CYP71E1 and NADPH-cytochrome P450 oxidoreductase from S. bicolor, efficient conversion of tyrosine to p-hydroxymandelonitrile was observed.     

Transgenic tobacco and Arabidopsis plants expressing the two multifunctional sorghum cytochrome P450 enzymes, CYP79A1 and CYP71E1, are cyanogenic and accumulate metabolites derived from intermediates in Dhurrin biosynthesis

Novel cyanogenic plants have been generated by the simultaneous expression of the two multifunctional sorghum (Sorghum bicolor [L.] Moench) cytochrome P450 enzymes CYP79A1 and CYP71E1 in tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis under the regulation of the constitutive 35S promoter. CYP79A1 and CYP71E1 catalyze the conversion of the parent amino acid tyrosine to p-hydroxymandelonitrile, the aglycone of the cyanogenic glucoside dhurrin. CYP79A1 catalyzes the conversion of tyrosine to p-hydroxyphenylacetaldoxime and CYP71E1, the subsequent conversion to p-hydroxymandelonitrile. p-Hydroxymandelonitrile is labile and dissociates into p-hydroxybenzaldehyde and hydrogen cyanide, the same products released from dhurrin upon cell disruption as a result of pest or herbivore attack. In transgenic plants expressing CYP79A1 as well as CYP71E1, the activity of CYP79A1 is higher than that of CYP71E1, resulting in the accumulation of several p-hydroxyphenylacetaldoxime-derived products in the addition to those derived from p-hydroxymandelonitrile. Transgenic tobacco and Arabidopsis plants expressing only CYP79A1 accumulate the same p-hydroxyphenylacetaldoxime-derived products as transgenic plants expressing both sorghum cytochrome P450 enzymes. In addition, the transgenic CYP79A1 Arabidopsis plants accumulate large amounts of p-hydroxybenzylglucosinolate. In transgenic Arabidopsis expressing CYP71E1, this enzyme and the enzymes of the pre-existing glucosinolate pathway compete for the p-hydroxyphenylacetaldoxime as substrate, resulting in the formation of small amounts of p-hydroxybenzylglucosinolate. Cyanogenic glucosides are phytoanticipins, and the present study demonstrates the feasibility of expressing cyanogenic compounds in new plant species by gene transfer technology to improve pest and disease resistance.     

Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803

Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66 mg/L of p-hydroxyphenylacetaldoxime and 5 mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3 mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation.     

Characterization and expression profile of two UDP-glucosyltransferases, UGT85K4 and UGT85K5, catalyzing the last step in cyanogenic glucoside biosynthesis in cassava

Manihot esculenta (cassava) contains two cyanogenic glucosides, linamarin and lotaustralin, biosynthesized from l ‐valine and l ‐isoleucine, respectively. In this study, cDNAs encoding two uridine diphosphate glycosyltransferase (UGT) paralogs, assigned the names UGT85K4 and UGT85K5, have been isolated from cassava. The paralogs display 96% amino acid identity, and belong to a family containing cyanogenic glucoside‐specific UGTs from Sorghum bicolor and Prunus dulcis. Recombinant UGT85K4 and UGT85K5 produced in Escherichia coli were able to glucosylate acetone cyanohydrin and 2‐hydroxy‐2‐methylbutyronitrile, forming linamarin and lotaustralin. UGT85K4 and UGT85K5 show broad in vitro substrate specificity, as documented by their ability to glucosylate other hydroxynitriles, some flavonoids and simple alcohols. Immunolocalization studies indicated that UGT85K4 and UGT85K5 co‐occur with CYP79D1/D2 and CYP71E7 paralogs, which catalyze earlier steps in cyanogenic glucoside synthesis in cassava. These enzymes are all found in mesophyll and xylem parenchyma cells in the first unfolded cassava leaf. In situ PCR showed that UGT85K4 and UGT85K5 are co‐expressed with CYP79D1 and both CYP71E7 paralogs in the cortex, xylem and phloem parenchyma, and in specific cells in the endodermis of the petiole of the first unfolded leaf. Based on the data obtained, UGT85K4 and UGT85K5 are concluded to be the UGTs catalyzing in planta synthesis of cyanogenic glucosides. The localization of the biosynthetic enzymes suggests that cyanogenic glucosides may play a role in both defense reactions and in fine‐tuning nitrogen assimilation in cassava.     

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.     

A combined biochemical screen and TILLING approach identifies mutations in Sorghum bicolor L. Moench resulting in acyanogenic forage production

Cyanogenic glucosides are present in several crop plants and can pose a significant problem for human and animal consumption, because of their ability to release toxic hydrogen cyanide. Sorghum bicolor L. contains the cyanogenic glucoside dhurrin. A qualitative biochemical screen of the M2 population derived from EMS treatment of sorghum seeds, followed by the reverse genetic technique of Targeted Induced Local Lesions in Genomes (TILLING), was employed to identify mutants with altered hydrogen cyanide potential (HCNp). Characterization of these plants identified mutations affecting the function or expression of dhurrin biosynthesis enzymes, and the ability of plants to catabolise dhurrin. The main focus in this study is on acyanogenic or low cyanide releasing lines that contain mutations in CYP79A1, the cytochrome P450 enzyme catalysing the first committed step in dhurrin synthesis. Molecular modelling supports the measured effects on CYP79A1 activity in the mutant lines. Plants harbouring a P414L mutation in CYP79A1 are acyanogenic when homozygous for this mutation and are phenotypically normal, except for slightly slower growth at early seedling stage. Detailed biochemical analyses demonstrate that the enzyme is present in wild-type amounts but is catalytically inactive. Additional mutants capable of producing dhurrin at normal levels in young seedlings but with negligible leaf dhurrin levels in mature plants were also identified. No mutations were detected in the coding sequence of dhurrin biosynthetic genes in this second group of mutants, which are as tall or taller, and leafier than nonmutated lines. These sorghum mutants with reduced or negligible dhurrin content may be ideally suited for forage production.     

Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin. Cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes

The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes L-valine as well as L-isoleucine consistent with the co-occurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k(c) value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.     

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.     

Biosynthesis of cyanogenic glucosides in Triglochin maritima and the involvement of cytochrome P450 enzymes.

The biosynthesis of the two cyanogenic glucosides, taxiphyllin and triglochinin, in Triglochin maritima (seaside arrow grass) has been studied using undialyzed microsomal preparations from flowers and fruits. Tyrosine was converted to p-hydroxymandelonitrile with V(max) and K(m) values of 36 nmol mg(-1) g(-1) fresh weight and 0.14 mM, respectively. p-Hydroxyphenylacetaldoxime and p-hydroxyphenylacetonitrile accumulated as intermediates in the reaction mixtures. Using radiolabeled tyrosine as substrate, the radiolabel was easily trapped in p-hydroxyphenylacetaldoxime and p-hydroxyphenylacetonitrile when these were added as unlabeled compounds. p-Hydroxyphenylacetaldoxime was the only product obtained using microsomes prepared from green leaves or dialyzed microsomes prepared from flowers and fruits. These data contrast earlier reports (H?sel and Nahrstedt, Arch. Biochem. Biophys. 203, 753-757, 1980; and Cutler et al., J. Biol. Chem. 256, 4253-4258, 1981) where p-hydroxyphenylacetaldoxime was found not to accumulate. All steps in the conversion of tyrosine to p-hydroxymandelonitrile were found to be catalyzed by cytochrome P450 enzymes as documented by photoreversible carbon monoxide inhibition, inhibition by antibodies toward NADPH-cytochrome P450 oxidoreductase, and by cytochrome P450 inhibitors. We hypothesize that cyanogenic glucoside synthesis in T. maritima is catalyzed by multifunctional cytochrome P450 enzymes similar to CYP79A1 and CYP71E1 in Sorghum bicolor except that the homolog to CYP71E1 in T. maritima exhibits a less tight binding of p-hydroxyphenylacetonitrile, thus permitting the release of this intermediate and its conversion into triglochinin.     

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.     

The bifurcation of the cyanogenic glucoside and glucosinolate biosynthetic pathways

The biosynthetic pathway for the cyanogenic glucoside dhurrin in sorghum has previously been shown to involve the sequential production of (E)‐ and (Z)‐p‐hydroxyphenylacetaldoxime. In this study we used microsomes prepared from wild‐type and mutant sorghum or transiently transformed Nicotiana benthamiana to demonstrate that CYP79A1 catalyzes conversion of tyrosine to (E)‐p‐hydroxyphenylacetaldoxime whereas CYP71E1 catalyzes conversion of (E)‐p‐hydroxyphenylacetaldoxime into the corresponding geometrical Z‐isomer as required for its dehydration into a nitrile, the next intermediate in cyanogenic glucoside synthesis. Glucosinolate biosynthesis is also initiated by the action of a CYP79 family enzyme, but the next enzyme involved belongs to the CYP83 family. We demonstrate that CYP83B1 from Arabidopsis thaliana cannot convert the (E)‐p‐hydroxyphenylacetaldoxime to the (Z)‐isomer, which blocks the route towards cyanogenic glucoside synthesis. Instead CYP83B1 catalyzes the conversion of the (E)‐p‐hydroxyphenylacetaldoxime into an S‐alkyl‐thiohydroximate with retention of the configuration of the E‐oxime intermediate in the final glucosinolate core structure. Numerous microbial plant pathogens are able to detoxify Z‐oximes but not E‐oximes. The CYP79‐derived E‐oximes may play an important role in plant defense.