The biosynthesis of cyanogenic glucosides in seedlings of cassava (Manihot esculenta Crantz)

Koch, Birgit; Nielsen, Vibeke Skovgaard; Halkier, Barbara Ann; Olsen, Carl Erik; Møller, Birger Lindberg

doi:10.1016/0003-9861(92)90062-2

Cited by 86 publications

(49 citation statements)

References 32 publications

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“…The substrate specificity of CYP79A2 seems to be rather narrow, as neither L-tyrosine, DL-homophenylalanine, L-tryptophan, nor L-methionine are metabolized by the enzyme, even though the former two amino acids are structurally similar to phenylalanine and function as precursors of glucosinolates. The high substrate specificity is in agreement with results obtained with CYP79 homologues involved in the biosynthesis of cyanogenic glucosides (23,29,44). The kinetic data for recombinant E. coli does not contain endogenous cytochromes P450 or NADPH:cytochrome P450 reductase.…”

Section: Fig 2 N-terminal Amino Acid Sequences Deduced From the Cypsupporting

confidence: 87%

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

Wittstock¹,

Halkier²

2000

Journal of Biological Chemistry

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235

161

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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 Nhydroxylase 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 mol liter ؊1 for L-phenylalanine. Neither L-tyrosine, L-tryptophan, Lmethionine, 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.Glucosinolates are amino acid-derived, secondary plant products containing a sulfate and a thioglucose moiety. Glucosinolates are found throughout the order Capparales, which includes agriculturally important crops of the Brassicaceae family such as oilseed rape and Brassica forages and vegetables, and the model plant Arabidopsis thaliana L. Upon tissue damage, glucosinolates are rapidly hydrolyzed to biologically active degradation products by the thioglucosidase myrosinase (EC 3.2.3

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Section: Fig 2 N-terminal Amino Acid Sequences Deduced From the Cypsupporting

confidence: 87%

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

Wittstock¹,

Halkier²

2000

Journal of Biological Chemistry

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235

161

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“…7). We have previously reported that the tegument in cassava seeds contains a potent Cyt P450 inhibitor (Koch et al, 1992) and that this also applies to the seed coat of sorghum (Halkier and Møller, 1989). The physiological role of this inhibitory activity is currently not understood but may serve to block detrimental production of cyanogenic compounds that would end up being degraded with concomitant release of HCN when the tegument dries out during the final stages of fruit ripening.…”

Section: Discussionmentioning

confidence: 99%

Bitterness in Almonds

et al. 2008

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Bitterness in almond (Prunus dulcis) is determined by the content of the cyanogenic diglucoside amygdalin. The ability to synthesize and degrade prunasin and amygdalin in the almond kernel was studied throughout the growth season using four different genotypes for bitterness. Liquid chromatography-mass spectrometry analyses showed a specific developmentally dependent accumulation of prunasin in the tegument of the bitter genotype. The prunasin level decreased concomitant with the initiation of amygdalin accumulation in the cotyledons of the bitter genotype. By administration of radiolabeled phenylalanine, the tegument was identified as a specific site of synthesis of prunasin in all four genotypes. A major difference between sweet and bitter genotypes was observed upon staining of thin sections of teguments and cotyledons for b-glucosidase activity using Fast Blue BB salt. In the sweet genotype, the inner epidermis in the tegument facing the nucellus was rich in cytoplasmic and vacuolar localized b-glucosidase activity, whereas in the bitter cultivar, the b-glucosidase activity in this cell layer was low. These combined data show that in the bitter genotype, prunasin synthesized in the tegument is transported into the cotyledon via the transfer cells and converted into amygdalin in the developing almond seed, whereas in the sweet genotype, amygdalin formation is prevented because the prunasin is degraded upon passage of the b-glucosidaserich cell layer in the inner epidermis of the tegument. The prunasin turnover may offer a buffer supply of ammonia, aspartic acid, and asparagine enabling the plants to balance the supply of nitrogen to the developing cotyledons.

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“…Although both enzymes possessed high K m values ranging from ;0.3 to 3 mM (Table 1), they were apparently able to accept all substrates in planta also since the various aldoxime products were detected both in herbivore-induced P. trichocarpa (Figures 1 and 6; see Supplemental Table 5 online) as well as in N. benthamiana overexpressing the two CYP79 genes (Figure 4; see Supplemental Tables 2 and 3 online). High K m values for amino acid substrates have been reported for other CYP79 enzymes (Koch et al, 1992;Halkier et al, 1995;Andersen et al, 2000;Forslund et al, 2004), and it has been suggested that low substrate affinity has evolved to avoid possible depletion of the free amino acid pool in plants (Andersen et al, 2000).…”

Section: Cyp79d6v3 and Cyp79d7v2 Have Broad Substrate Specificity Andmentioning

confidence: 98%

“…The CYP79s functioning in cyanogenic glucoside and glucosinolate biosynthesis are entry-level enzymes that usually possess high specificities for their amino acid substrates and so determine the specificity of the entire pathway (Koch et al, 1992;Andersen et al, 2000;Wittstock and Halkier, 2000;Forslund et al, 2004). By contrast, recombinant CYP79D6v3 and CYP79D7v2 from P. trichocarpa each accepted a variety of different amino acids as substrates, converting them into the corresponding aldoximes (Figure 3).…”

Section: Cyp79d6v3 and Cyp79d7v2 Have Broad Substrate Specificity Andmentioning

confidence: 99%

Two Herbivore-Induced Cytochrome P450 Enzymes CYP79D6 and CYP79D7 Catalyze the Formation of Volatile Aldoximes Involved in Poplar Defense

et al. 2013

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Aldoximes are known as floral and vegetative plant volatiles but also as biosynthetic intermediates for other plant defense compounds. While the cytochrome P450 monooxygenases (CYP) from the CYP79 family forming aldoximes as biosynthetic intermediates have been intensively studied, little is known about the enzymology of volatile aldoxime formation. We characterized two P450 enzymes, CYP79D6v3 and CYP79D7v2, which are involved in herbivore-induced aldoxime formation in western balsam poplar (Populus trichocarpa). Heterologous expression in Saccharomyces cerevisiae revealed that both enzymes produce a mixture of different aldoximes. Knockdown lines of CYP79D6/7 in gray poplar (Populus 3 canescens) exhibited a decreased emission of aldoximes, nitriles, and alcohols, emphasizing that the CYP79s catalyze the first step in the formation of a complex volatile blend. Aldoxime emission was found to be restricted to herbivore-damaged leaves and is closely correlated with CYP79D6 and CYP79D7 gene expression. The semi-volatile phenylacetaldoxime decreased survival and weight gain of gypsy moth (Lymantria dispar) caterpillars, suggesting that aldoximes may be involved in direct defense. The wide distribution of volatile aldoximes throughout the plant kingdom and the presence of CYP79 genes in all sequenced genomes of angiosperms suggest that volatile formation mediated by CYP79s is a general phenomenon in the plant kingdom.

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The biosynthesis of cyanogenic glucosides in seedlings of cassava (Manihot esculenta Crantz)

Cited by 86 publications

References 32 publications

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

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

Bitterness in Almonds

Two Herbivore-Induced Cytochrome P450 Enzymes CYP79D6 and CYP79D7 Catalyze the Formation of Volatile Aldoximes Involved in Poplar Defense

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