Biosynthesis and Degradation of Cyanogenic Glycosides

1.31 Biosynthesis and Degradation of Cyanogenic Glycosides MONICA A. HUGHES University of Newcastle upon Tyne, UK 0[20[0 INTRODUCTION 0[20[0[0 0[20[0[1 0[20[0[2 0[20[0[3 0[20[0[4

770

Cyano`enesis Phylo`enic Distribution of Cyano`enesis in Plants Cyano`enesis in PlantÐAnimal and PlantÐMicrobe Interactions Cyano`enesis and Metabolism Compartmentation

0[20[1 CHEMICAL NATURE OF CYANOGENIC GLYCOSIDES 0[20[1[0 0[20[1[1 0[20[1[2 0[20[1[3 0[20[1[4 0[20[1[5

Precursor Amino Acids and Nicotinic Acid Cyano`enic Glycosides Derived from Valine and Isoleucine Cyano`enic Glycosides Derived from Leucine Cyano`enic Glycosides Derived from Phenylalanine Cyano`enic Glycosides Derived from Tyrosine Cyano`enic Glycosides Derived from the Non!protein Amino Acid "1!Cyclopentenyl#`lycine and Nicotinic Acid

0[20[2 BIOSYNTHESIS OF CYANOGENIC GLYCOSIDES

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0[20[2[0 Cyanohydrin "a!Hydroxynitrile# Biosynthesis 0[20[2[1 UDP!`lucose Glucosyltransferase 0[20[3 DEGRADATION OF CYANOGENIC GLYCOSIDES 0[20[3[0 b!Glycosidases 0[20[3[1 a!Hydroxynitrile Lyases

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0[20[4 REFERENCES

782

0[20[0 INTRODUCTION 0[20[0[0 Cyanogenesis The term cyanogenesis describes the release of hydrogen cyanide "HCN# from damaged plant "and some insect0# tissue[ Although HCN can occur in small quantities in all plant tissues\ for example cyanide is a product of ethene biosynthesis\1 in cyanogenic species large quantities of HCN are produced only following tissue disruption[ Cyanogenesis was _rst described in plants in 0792 and has now been reported in at least 1599 species\ within 029 di}erent families[2\3 In approximately 364 of these species\ the source of HCN has been identi_ed3 and shown to result from the enzymatic degradation of cyanogenic glycosides[ In most species this degradation involves hydrolysis by one or more b!glycosidases\ followed by enzymatic breakdown of the aglycone "cyanohydrin# to a carbonyl compound and HCN by an a!hydroxynitrile lyase[ 770

771

Biosynthesis and De`radation of Cyano`enic Glycosides

The last general review of cyanogenesis was published in 08894 and since that time there have been major advances in our understanding of the biosynthesis of cyanoglycosides5\6 and the crystal structure of two of the enzymes responsible for the degradation of cyanogenic glycosides has been solved[7\8

0[20[0[1 Phylogenic Distribution of Cyanogenesis in Plants Angiosperm families that are noted for cyanogenesis are Rosaceae "049 species#\ Leguminosae "014 species#\ Gramineae "099 species#\ Araceae "49 species#\ Euphorbiaceae "49 species#\ Compositae "49 species#\ and Passi~oraceae "29 species#[09 In addition\ some gymnosperms "for example\ Taxus baccata L[00# and ferns "for example\ Davallia trichomanoides01# are cyanogenic[ In many plant species only a single cyanogenic glycoside has been reported\ however\ in an increasing number of plants\ more than one compound has been found[ In barley "Hordeum vul`are L[#\ for example\ _ve cyanogenic glycosides have been identi_ed in the leaf epidermal cells[02 In barley\ these compounds di}er in the structure of the aglycone but in other species cyanogenic glycosides with di}erent sugar residues are found[ For example\ both monoglucosides and diglucosides are found in Prunus serotina Ehrh[ "black cherry#03 and in Linum usitatissimum L[ "~ax#[04 Cyanogenesis is of limited use in angiosperm phylogeny studies because it occurs in both primitive and advanced groups and because the occurrence is erratic in most families and even within some genera "Trifolium#[05 An interesting situation is found in the genus Acacia\ where cyclic cyanogenic glucosides are found in Australian species whereas aliphatic groups are found in African\ Asian\ and American species[05 Some plant species are polymorphic for the cyanogenic phenotype\ that is both cyanogenic and acyanogenic plants may occur in the same species[ The most extensive studies of the cyanogenic polymorphism have been carried out in the herbage legume\ Trifolium repens L[ "white clover# "reviewed by Hughes06# and in Lotus corniculatus L[ "birds|s foot trefoil#[07 The cyanogenic poly! morphism in white clover is controlled by alleles of two independently segregating loci "Li and Ac#[ It shows diploid inheritance and only plants which contain a functional\ dominant allele of both loci are cyanogenic[ In two papers published in 0843\ Daday08\19 demonstrated a clear association between the frequency of the cyanogenic morph in natural populations of white clover and the mean January isotherm\ such that populations at higher altitudes and higher latitudes have lower frequencies of cyanogenic plants[ This association has been con_rmed by other workers and a survey of the US white clover germplasm collection has shown that accessions from low altitudes and from sites with a high winter temperature\ lower summer precipitation\ spring sunshine\ and snow cover\ have higher frequencies of cyanogenic plants[10 The cyanogenic polymorphism in white clover is thought to be maintained by selection for the acyanogenic morph by increased frost damage in cyanogenic plants and a balancing selection for the cyanogenic morph\ caused by increased predation of acyanogenic plants by small predators "see Section 0[20[0[2#[06 Quantitative variation in levels of cyanogenesis has also been documented and\ for example\ variation in levels of cyanogenic glucoside in mature plants of the tropical species Turnera ulmifolia L[\ collected from di}erent locations in Jamaica\ has been shown to have a genetic basis\11 however\ the selective agents have not been _rmly identi_ed[ Variation in levels of cyanogenesis has also been reported for single plants\ depending upon both external environmental factors and development[ In white clover cyanogenic glucoside synthesis is in~uenced by temperature12 and\ in cassava "Manihot esculenta Crantz# leaves\ diurnal variation in cyanogenic glucoside levels has been reported[13 Patterns of changes in cyanogenic glucoside content during development vary between species[ In Hevea species\ the seeds contain high levels of cyano! genic glucosides and as the seeds germinate levels in the endosperm fall but levels in the embryo: plantlet increase14 "see Section 0[20[0[3#[ In cassava\ which is also a member of the Euphorbiaceae and produces the same cyanogenic glucosides as Hevea "linamarin "0# and lotaustralin "1#\ see Section 0[20[1#\ the seeds contain virtually no cyanogenic glucoside[ Both cyanogenic glucosides\ and the enzymes responsible for cyanogenic glucoside degradation during cyanogenesis\ are synthesized rapidly de novo during germination in cassava "Figure 0#[

0[20[0[2 Cyanogenesis in PlantÐAnimal and PlantÐMicrobe Interactions The toxicity of hydrogen cyanide to insects is well known and cyanogenesis is widely regarded as a defense mechanism which has evolved because it protects plants from predation by small herbivores^

Biosynthesis and De`radation of Cyano`enic Glycosides

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Figure 0 Production of cyanogenic glucosides and the degrading enzymes "b!D!glucosidase and a!hydroxy! nitrile lyase# in di}erent organs during germination of Manihot esculenta Crantz "cassava# seeds[

however\ the interaction of cyanogenic host plant and herbivore is complicated in many cases by co!evolution[ In species which are polymorphic for cyanogenesis\ there is abundant evidence from both natural habitats and experimental data that selective grazing of the acyanogenic form occurs^06\07\15 however\ there is considerable variation between species of herbivores in selective feeding behaviour[ In plant species such as sorghum "Sor`hum bicolor L[ Moench[# and cassava\ which are not polymorphic for cyanogenesis\ there is quantitative variation in the levels of cyanide produced[ Studies of herbivore grazing in these species have compared feeding:damage in cultivars with di}erent levels of cyanide production "cyanogenic potential#[ Cassava is a crop which evolved in South America and although it appears to be resistant to many pests there are several specialized pests\ such as the hornworm "Erinnyis ello# and the green mite "Mononychellus tanajoa#\ which have co!evolved with cassava and show no preference between plants that are highly cyanogenic or not[16 The burrowing bug Cyrtomenus ber`i\ however\ appears to be a recent pest of the cassava crop in Colombia and laboratory and _eld experiments show that root damage from this pest is reduced in those cassava cultivars with a high cyanogenic potential[16 A number of herbivorous insect species are also cyanogenic0 and these may feed upon cyanogenic plants[ The larvae of Acraea horta "Lepidoptera# feed upon the leaves of the cyanogenic species Ki``elaria africana L[\ and the cyanogenic glucoside "gynocardin "06## which is taken up by the larvae\ is sequestered by the insect[17 However\ although some Zy`aena species eat birds| foot trefoil\ de novo synthesis of cyanogenic glucosides "linamarin "0# and lotaustralin "1## has been shown to occur in this genus of Lepidoptera[0\18 T[ repens L[ "white clover# is one of the primary host plants of the sulfur butter~y "Colias erate polio`raphys# and cyanogenic glucosides have been shown to serve as synergistic oviposition stimulants for this insect\ suggesting that they play a positive role in host selection[29

773

Biosynthesis and De`radation of Cyano`enic Glycosides

Many plant pathogens have the ability to detoxify the HCN produced from cyanogenic glycosides during cyanogenesis[ In only a small number of examples do high levels of cyanogenesis correlate with resistance to pathogens[ Lehman et al[20 report that infection of white clover by Sclerotina trifoliorum is reduced in the highly cyanogenic cultivar\ Arau[ However\ Lieberei et al[21 have shown that cyanogenesis inhibits active pathogen defence in plants and Microcyclus ulei\ which causes blight of Hevea brasiliensis Muell[ Arg[ "rubber tree#\ is not only tolerant of HCN but grows better in an HCN!containing atmosphere[22 This means that weakly cyanogenic plants may generally show more resistance to the pathogen than highly cyanogenic plants[

0[20[0[3 Cyanogenesis and Metabolism Although it is widely accepted that the cyanogenic system is a plant mechanism for protection against herbivores\ two other roles have been suggested\ namely that cyanogenic glycosides are either "i# waste products or "ii# intermediates in nitrogen metabolism[07 The hypothesis that cyanogenic glycosides are nitrogenous waste compounds is di.cult to defend[ Nitrogen is often limiting for plant growth and cyanogenic species are generally not limited to ecological habitats with nitrogen!rich soil[ Further\ other examples of nitrogenous waste products are not known in plants[ The presence of cyanogenic and acyanogenic individuals in polymorphic species\ such as white clover\ argues against a role in primary metabolism\ in these species at least[ Although cyanogenic glycosides do not have an essential role in primary metabolism and despite the general observation that they are stable compounds stored in cellular compartments that lack degrading b!glycosidases\ a number of examples exist where turnover of cyanogenic glycosides has been reported[ It has been suggested that cyanogenic diglucosides are metabolites of cyanogenic monoglucosides\ which can be translocated within the plant because they are resistant to the abundant monoglucosidase enzyme[ In seeds of H[ brasiliensis Muell[ Arg[\ the cyanogenic monoglucoside linamarin "0# accumulates in the endosperm[ After the onset of germination\ the levels of this glucoside in the endosperm decrease\ with a concomitant increase in the level of the diglucoside linustatin "3# in endosperm exudates[23 It is proposed that during germination the stored monoglucoside linamarin "0# is glycosylated to the diglucoside linustatin "3#[ This makes it resistant to the abundant apoplastic monoglucosidase so that it can be transported from the endosperm to the growing seedling\ where it is cleaved by a diglucosidase to produce HCN[ Negligible amounts of gaseous HCN are produced because cyanide is reassimilated into noncyanogenic compounds[23 Detoxi_cation of HCN to asparagine by b! cyanoalanine synthase produced in developing Hevea seedlings allows the HCN to reenter general metabolic pools[23 The monoglucoside linamarin "0#\ produced in the developing plantlet is syn! thesized de novo from the precursor amino acid\ valine "see Sections 0[20[1[1 and 0[20[2#[ The diglucoside linustatin "3# has also been isolated from ~ax\24 which also produces cyanogenic seeds\ suggesting that this mechanism may be general in plants[ In fact\ small amounts of dhurrin 5?!glucoside "the diglucoside produced by further glycosylation of the monoglucoside dhurrin "03# have been identi_ed in guttation droplets of S[ bicolor "L[# Moench[ seedlings\25 although this species does not have cyanogenic seeds[ Turnover of dhurrin "03# in green sorghum seedlings has also been demonstrated using an inhibitor of tyrosine "the precursor amino acid of dhurrin "03#\ see Section 0[20[1[4# biosynthesis and radiolabeled tyrosine in in vivo feeding experiments[26

0[20[0[4 Compartmentation Consistent with a role in plant defense\ cyanogenic glucosides are stored and separated from the catabolic enzymes in the intact plant by compartmentation at either tissue or subcellular levels[ Information about compartmentalization is not available for many cyanogenic species but it is clear\ from those which have been studied\ that the details of compartmentation di}er between species[ In the leaves of sorghum seedlings\ the cyanogenic glucoside is sequestered within the vacuoles of epidermal cells\ whereas the two degrading enzymes\ b!glucosidase and a!hydroxynitrile lyase\ are present almost exclusively in the underlying mesophyll cells\ within the chloroplasts and cytosol

Biosynthesis and De`radation of Cyano`enic Glycosides

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respectively[27\28 Large!scale hydrolysis of the cyanogenic glucoside can therefore only occur fol! lowing tissue disruption\ such as during herbivore attack[ Cassava is a member of the Euphorbiaceae and contains a network of latex vessels which run throughout the plant[ The number of vessels in di}erent organs and in di}erent tissues within organs varies[ These vessels are\ for example\ abundant in young leaf spongy mesophyll tissue but relatively rare in parenchyma of the swollen roots[ White et al[39 have demonstrated the presence of the cyanogenic glucoside\ linamarin "0#\ in cassava leaf vacuoles[ The _rst degrading enzyme\ a cyano! genic b!glucosidase with the trivial name linamarase "see Section 0[20[3[0#\ is primarily located in the latex vessels[30\31 The exact location of the a!hydroxynitrile lyase is not known but the structure of the protein "having no signal sequence or organelle retention signals# suggests that it is cytosolic[32 In white clover\ which produces the same cyanogenic glucosides as cassava but is a legume and therefore possesses no latex vessel system\ the cyanogenic b!glucosidase "see Section 0[20[3[0# has been shown to be apoplastic\ possibly present in cell walls[33 The techniques which are commonly used to demonstrate an apoplastic location for proteins34 are di.cult to interpret in a species with latex vessels containing latex under pressure\ and a number of reports of the apoplastic location of the cassava linamarase have to be interpreted with caution\ particularly since a latex control enzyme such as chitinase was not included in the experiments[35 Cyanogenesis in black cherry "P[ serotina# has been extensively studied by Poulton|s group[36Ð40 The kernels of black cherry seeds contain large quantities of the cyanogenic diglucoside "R#!amygdalin "01# and three catabolic enzymes] the diglucosidase amygdalin hydrolase^ the mono! glucosidase\ prunasin hydrolase^ and an a!hydroxynitrile lyase\ "R#!"¦#!mandelonitrile lyase[ These enzymes _rst appear in the seeds about 5 weeks after ~owering[ The two b!glucosidases are restricted to protein bodies in the procambium\ whereas the hydroxynitrile lyase occurs primarily in protein bodies in the cotyledonary parenchyma cells\ which is also the location of the cyanogenic diglucoside\ amygdalin "01#[ Thus\ in black cherry\ cyanogenesis in intact tissues of the developing seed is prevented by segregation of the _rst degrading enzyme\ amygdalin hydrolase\ and amygdalin "01# in di}erent tissues[

0[20[1 CHEMICAL NATURE OF CYANOGENIC GLYCOSIDES 0[20[1[0 Precursor Amino Acids and Nicotinic Acid Cyanogenic glycosides are of intermediate polarity\ being water!soluble compounds which are typically O!b!glycosides of a!hydroxynitriles "cyanohydrins#\ and are themselves relatively nontoxic to most organisms[ All of the 46 known higher plant cyanogenic glycosides are probably derived from the _ve hydrophobic L!amino acids\ valine\ isoleucine\ leucine\ phenylalanine and tyrosine\ the nonprotein amino acid "1!cyclopentenyl#glycine\ and nicotinic acid[3\25 Glucose is the sugar directly attached to the hydroxy of the cyanohydrin[ In addition to the optically active centres of the sugars\ the carbon of the cyanohydrin which is attached to the sugar\ the nitrile group\ and the hydrogen are also usually chiral[ Thus "R#! and "S#!epimers are known for most series and in some plants both epimers can co!occur\ for example\ "R#!prunasin "09# and "S#!sambunigrin "00# in Acacia species[41 The structures of cyanogenic glycosides and related compounds have been reviewed by Seigler\3 where all of their structures can be found[

0[20[1[1 Cyanogenic Glycosides Derived from Valine and Isoleucine The cyanogenic glucosides linamarin "0# and lotaustralin "1#\ which are derived from the amino acids valine and isoleucine\ respectively\ commonly co!occur\ although the proportion of each may vary[ Thus\ in cassava 84) of the total cyanogenic glucoside is linamarin "0#\42 whereas in white clover they are more or less equally abundant[43 These cyanogenic glycosides are recorded in more plant species than any of the other cyanogenic glycosides[ The "S#!epimer of lotaustralin\ "S#! epilotaustralin "2#\ is di.cult to distinguish from "R#!lotaustralin "1# and most reports do not specify which epimer is produced[ The diglucosides of linamarin "0# and lotaustralin "1# "linustatin "3# and neolinustatin "4# respectively# have been found at low levels in ~ax\24 Hevea\23 and cassava\44 all of which produce linamarin "0# and lotaustralin "1#[

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Biosynthesis and De`radation of Cyano`enic Glycosides

O

HO HO

OH

OH

OH NC HO HO

O

NC

NC O

OH

(R)-Lotaustralin (2)

(S)-Epilotaustralin (3) OH

OH HO HO

O

HO HO

O OH

OH Linamarin (1)

O

O OH

O

HO HO

O O

HO HO

OH

CN

O

O O

HO HO

O

CN

OH

OH Linustatin (4)

Neolinustatin (5)

0[20[1[2 Cyanogenic Glycosides Derived from Leucine Ten cyanogenic glycosides are known which have L!leucine as the precursor[ Four of these are illustrated ""5# to "8##[ "R#!Epiheterodendrin "8# is produced by germinating barley[ Its breakdown during fermentation of malted barley is a problem because the HCN released can react with ethanol to produce ethylcarbamate\ an established carcinogen[45 OH

OH H

O

HO HO

CN

O

HO HO

O

H

CN

O

OH

OH

(S)-Proacacipetalin (6)

(R)-Epiproacacipetalin (7)

OH

OH H

O

HO HO

CN

O

HO HO

O

H

CN

O

OH

OH

(S)-Heterodendrin (8)

(R)-Epiheterodendrin (9)

0[20[1[3 Cyanogenic Glycosides Derived from Phenylalanine One of the best!known cyanogenic glycosides\ "R#!amygdalin "01# is a diglucoside derived from phenylalanine[ This compound is commonly found in seeds of members of the Rosaceae\ such as black cherry\ almonds\ peaches\ and apricots[3 There are six monoglucosides known to be derived from phenylalanine\ including the monoglucoside equivalent of amygdalin\ "R#!prunasin "09#\ and the S!epimer of "R#!prunasin\ "S#!sambunigrin "00#[ OH HO HO

OH H

O

CN

O

HO HO

O OH

CN

OH

(R)-Prunasin (10)

(S)-Sambunigrin (11) OH

HO HO

H O

O O OH HO HO

H

O

O OH

(R)-Amygdalin (12)

CN

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Biosynthesis and De`radation of Cyano`enic Glycosides 0[20[1[4 Cyanogenic Glycosides Derived from Tyrosine

Seven cyanogenic glycosides are known which have tyrosine as a precursor[ This series also includes "R#! and "S#!epimers of monoglucosides and equivalent diglucosides[3\25 The structure of "R#!taxiphyllin "02# and "S#!dhurrin "03# are shown[ The biosynthesis of dhurrin "03# in etiolated sorghum seedlings is the best!understood biosynthetic system "see Section 0[20[2[0#[ OH

OH H

O

HO HO

CN OH

OH

H

O

HO HO

O

CN

O OH

OH

(R)-Taxiphyllin (13)

(S)-Dhurrin (14)

0[20[1[5 Cyanogenic Glycosides Derived from the Non!protein Amino Acid "1!Cyclopentenyl#glycine and Nicotinic Acid A group of 03 cyanogenic glycosides has been identi_ed\ probably derived from "1!cyclo! pentenyl#glycine\ three of which are illustrated "04\ 05\ 06#[ As with the other series\ the group includes R! and S!epimers and both mono! and diglucosides[ They are commonly found in Pas! si~oraceae[3 The compound acalyphin "07# from a member of the Euphorbiaceae "Acalypha indica L[# appears to be derived from nicotinic acid[46 CN

CN OH

O HO

O

OH O HO

OH

OH Deidaclin (15)

O

OH

OH Tetraphyllin A (16) OH

HO HO

O

OH

O

OH NC

OH H

OH Gynocardin (17)

HO HO

O

OMe NC O

OH HO

N

O

Me Acalyphin (18)

0[20[2 BIOSYNTHESIS OF CYANOGENIC GLYCOSIDES 0[20[2[0 Cyanohydrin "a!Hydroxynitrile# Biosynthesis The biosynthetic pathway of cyanogenic glycosides in higher plants is considered to be closely related to the pathways producing glucosinolates\ organic nitro! compounds and possibly nitrile glycosides[ In outline\ a membrane!bound enzyme system converts a precursor amino acid to an a!hydroxynitrile via an oxime[ The a!hydroxynitrile is then glucosylated by a soluble UDP!glucose glucosyltransferase[ Scheme 0 shows the biosynthetic pathway for the cyanogenic glucoside\ dhurrin\ in Sor`hum bicolor "L[# Moench[ The reactions catalyzed by cytochrome P349TYR are boxed[56 In vivo feeding of labeled amino acid precursors to plants which are actively synthesizing cyanogenic glycoside commonly results in extremely e.cient labeling of the cyanogenic glycosides\ re~ecting the very large quantities of these compounds which can accumulate in some tissues[ Early biosynthetic studies demonstrated the direct incorporation of the Cb0Ca0N moiety of the precursor amino acid into the cyanogenic glycoside\ indicating that all of the intermediates contain it[47 The carboxyl carbon of the amino acid is lost\ the a!carbon bearing the amine group is oxidized to the level of nitrile\ and the b!carbon is oxygenated to yield a hydroxy group\ which bears the glucose of the glucoside[ All early studies found negligible accumulation of pathway intermediates

777

Biosynthesis and De`radation of Cyano`enic Glycosides

HO

CH

CH2

CO2H

NH2 L-tyrosine NADPH + O2 NADP+

HO

CH

CH2

CO2H

NH OH N-hydroxytyrosine NADPH + O2 NADP+

HO

CH

CH2

CO2H

N

HO OH N,N-dihydroxytyrosine

HO

CH

CH2

CO2H

N O 2-nitroso-3-(p-hydroxyphenyl)propanoic acid

HO

CH2

CH N

OH

(E)-p-hydroxyphenylacetaldehyde oxime

HO

CH2

CH

HO

N

C N

CH2 p-hydroxyphenylacetonitrile

HO (Z)-p-hydroxyphenylacetaldehyde oxime

NADPH + O2 NADP+ in vitro

HO

CH OH p-hydroxymandelonitrile

C N

HO

CHO + HCN

p-hydroxybenzaldehyde in vivo

HO

CH O Dhurrin

Scheme 1

C N Glucose

Biosynthesis and De`radation of Cyano`enic Glycosides

778

and the pathway was therefore referred to as {channeled|[ To date\ the most detailed studies on cyanogenic glycoside biosynthesis have been carried out for dhurrin "03# in sorghum[ Based on the similarity of the biosynthetic reactions "when studied# in other cyanogenic plants\ it is believed that the information obtained in sorghum can be extrapolated to other plants[ In vitro\ biosynthetic studies were made possible by the isolation of a biosynthetically active microsomal preparation from etiolated seedlings of sorghum\ which was capable of converting L!tyrosine into "S#!p!hydroxymandelonitrile\ the a!hydroxynitrile "cyanohydrin# precursor of dhurrin "03#[48 Thus this preparation can carry out all except the _nal glycosylation step in the dhurrin biosynthetic pathway "Scheme 0#[ It has been demonstrated that the sorghum microsomal preparation can produce and metabolize the intermediates N!hydroxytyrosine\ 1!nitroso!2! "p!hydroxyphenyl#propanoic acid\ "E#! and "Z#!p!hydroxyphenylacetaldehyde oxime\ p!hydroxy! phenylacetonitrile and p!hydroxymandelonitrile\ which are shown in the biosynthetic pathway in Scheme 0[48Ð53 The compound N\N!dihydroxytyrosine is very labile and has not been isolated[ The only intermediate in this pathway that freely exchanges with exogenously supplied material is "Z#!p!hydroxyphenylacetaldehyde oxime[ Stoichiometric measurements of oxygen consumption and biosynthetic activity have shown that two molecules of oxygen are consumed in two consecutive N!hydroxylation reactions in the con! version of L!tyrosine to p!hydroxyphenylacetaldehyde oxime and one oxygen in the C!hydroxylation converting p!hydroxyphenylacetonitrile to p!hydroxymandelonitrile[53 The L!tyrosine N!hydroxylase is inhibited by carbon monoxide and this inhibition is reversed by 349 nm light[ This demonstrates that the enzyme is a cytochrome P349 dependent monooxygenase[54 A sorghum seedling heme!thiolate enzyme\ cytochrome P349TYR\ which catalyzes the conversion of L!tyrosine to p!hydroxyphenylacetaldehyde oxime\ has been isolated\ puri_ed\ and characterized[5\6 Cytochrome P349 dependent monooxygenase reactions are dependent on small electron transport chains\ where reducing equivalents from NADPH are transferred via a ~avin!containing oxi! doreductase to the terminal cytochrome P349[ When a reconstituted complex containing puri_ed cytochrome P349TYR\ NADPH!cytochrome P349 oxidoreductase\ and L!a!dilauroylphos! phatidylcholine is fed L!tyrosine in the presence of NADPH\ p!hydroxyphenylacetaldehyde oxime accumulates[6 A cDNA clone encoding the sorghum cytochrome P349TYR has been isolated and expressed in Escherichia coli[55\56 The puri_ed E[ coli recombinant protein also catalyzes the con! version of L!tyrosine to p!hydroxyphenylacetaldehyde oxime in reconstitution experiments\ using sorghum NADPHÐcytochrome P349 reductase[55 The surprising biosynthetic properties of this cytochrome P349 raise questions about the nature of the intermediates between amino acid and oxime\ shown in Scheme 0[ Could some of the compounds detected in earlier experiments represent arti_cially generated stable forms of transition states< Conversion of the p!hydroxyphenylacetaldehyde oxime to p!hydroxymandelonitrile in the pres! ence of oxygen\ by isolated sorghum microsomes\ involves a C!hydroxylation reaction of p!hydroxyphenylacetonitrile[ This reaction also shows inhibition by carbon monoxide that is reversed by 349 nm light\ characteristic of cytochrome P349^54 in addition\ the reaction is inhibited by antibodies to NADPH!cytochrome P349 oxidase[ It was not possible to dissect the cofactor requirements for the conversion of p!hydroxyphenylacetaldehyde oxime to p!hydroxymandelonitrile into two separate reactions and the intermediate p!hydroxyphenylacetonitrile does not accumulate in the reaction mixture[54 Puri_cation and characterization of the protein"s# responsible for oxime metabolism in sorghum have not been reported[ Microsomal preparations\ which can carry out metabolism of valine and isoleucine to produce linamarin "0# and lotaustralin "1#\ respectively\ have also been isolated from white clover\57 ~ax\58 and cassava[69 Further it has been shown that the metabolism of valine to linamarin and isoleucine to lotaustralin is carried out by the same proteins[60 In white clover\ plants possessing only non! functional ac alleles are unable to synthesize either linamarin or lotaustralin[61 In vivo and in vitro labeling experiments have shown that ac ac plants have at least two steps in the conversion of amino acids to a!hydroxynitrile missing from the microsomal preparations[ Thus microsomes from ac ac plants are "i# unable to produce the oxime intermediate and "ii# unable to convert fed oxime to a! hydroxynitrile\ whilst Ac Ac microsomes can carry out both steps[57 Microsomes have also been isolated from Tri`lochin maritima L[ which produce the cyanohydrin of taxiphyllin "02# from tyrosine[62 It is a general feature of these biosynthetic studies that the oxime is the only intermediate which can be easily detected or used in the cyanohydrin product\ either when fed in vivo or incorporated into the microsome reaction mix[ It is tempting to speculate that in all higher plant species\ the metabolism of amino acid to cyanohydrin involves just two cytochrome P349 enzymes with the oxime being the only true intermediate[

789

Biosynthesis and De`radation of Cyano`enic Glycosides

0[20[2[1 UDP!glucose Glucosyltransferase The _nal step in the biosynthesis of cyanogenic glycosides is glucosylation of the a!hydroxylnitrile "see Scheme 0#[ UDP!glucose glucosyltransferase enzymes\ which are capable of glucosylating the respective a!hydroxylnitrile\ have been partially puri_ed from black cherry\63 T[ maritima L[\64 ~ax\65 sorghum\66 and cassava[67 All of the enzymes behave as soluble enzymes and are not found associated with the microsomal preparations which synthesize the a!hydroxynitrile[ The soluble nature of these glucosyltransferases is perhaps surprising given "i# the {channeled| nature of a!hydroxynitrile biosynthesis by membrane!bound "microsomal# enzymes\ "ii# the instability of the a!hydroxynitriles\ and "iii# the localization of the cyanogenic glycosides in vacuoles[ Glycosylation of a number of secondary plant compounds\ including ~avonoids and steroidal alkaloids\ occurs at the end of their biosynthetic pathway[ The most common sugar is glucose and these reactions are also catalyzed by a UDP!glucose glucosyltransferase to produce stable water! soluble compounds which are often transported into the vacuole[ Given the reported speci_city of these enzymes and the large number of potential substrates\ a wide range of di}erent glucosyl! transferases may be expected to occur within a single plant species[68 Further\ considerable deduced amino acid sequence homology exists between those plant glucosyltransferases which have been cloned[79 These factors have contributed to the di.culty of purifying and characterizing the cyano! genic glycoside UDP!glucose glucosyltransferase and detailed information about these proteins does not exist[

0[20[3 DEGRADATION OF CYANOGENIC GLYCOSIDES 0[20[3[0 b!Glycosidases Scheme 1 shows the degradation of linamarin "0# and lotaustralin "1# by b!D!glucosidase and a!hydroxynitrile lyase[ The _rst step in the degradation of cyanogenic glycosides is hydrolysis by one or more b!glycosidases[ In most cyanogenic plant species one or more b!glycosidases are produced which have pronounced speci_city for their endogenous cyanogenic glycoside"s#^70 however\ examples of enzymes with very broad speci_city exist "e[g[\ ~ax71#[ All of the cyanogenic b!glycosidases tested will hydrolyze the synthetic substrates\ p!nitrophenyl!b!D!glucoside and p! nitrophenyl!b!D!galactoside\ and are therefore not entirely speci_c for either the aglycone or the sugar moiety of the substrate[ The cyanogenic b!glycosidases that have been investigated are all glycoproteins with a subunit molecular mass of 44Ð54×092\ isoelectric points between pH 3[9Ð4[4\ and acidic pH optima "pH 3[9Ð5[1#[4 R

CN O

H2O

gluc

R = H Linamarin (1) R = Me Lotaustralin (2)

β-glucosidase

R

CN OH

α-Hydroxynitrile

α-hydroxynitrile lyase

HCN

+

R

O

Propanone or Butanone

Scheme 2

Hydrolysis of cyanogenic disaccharides may be either {simultaneous|\ such as linustatin"3# in H[ brasiliensis Muell[ Arg[72 and vicianin inVicia au`ustifolia L[\73 where hydrolysis yields a disaccharide plus aglycone[ Alternatively hydrolysis can be sequential\ where two hydrolytic reactions are cat! alyzed by two separate b!glycosidases[ The best documented example of sequential hydrolysis is amygdalin "01# hydrolysis in black cherry[74 Amygdalin hydrolase degrades the cyanogenic diglu! coside\ amygdalin "01#\ to produce glucose and the cyanogenic monoglucoside\ prunasin"09#[ Pru! nasin is subsequently hydrolyzed by prunasin hydrolase to produce glucose and the a!hydroxynitrile\ mandelonitrile[ Four isozymes of amygdalin hydrolase and three isozymes of prunasin hydrolase have been puri_ed from black cherry[74 The cyanogenic b!glucosidase responsible for hydrolysis of the monoglucosides\ linamarin "0# and lotaustralin "1#\ has the trivial name\ linamarase[ This enzyme has been cloned as cDNA from white clover75 and from cassava[76 Classi_cation of these enzymes on the basis of amino acid sequence similarity77\78 places them in Family 0 of the glycosyl hydrolases[ These are known as retaining glycosidases due to retention of the con_guration of the anomeric centre of the substrate during hydrolysis in a double displacement mechanism89 "Scheme 2#[ Stereoselective substitution in

Biosynthesis and De`radation of Cyano`enic Glycosides

780

this position\ by water or another nucleophile\ is supported by a catalytic dyad composed of an acid catalyst residue acting in the departure of the aglycone from the substrate[ A nucleophile group "generally a carboxylate# stabilizes the oxocarbonium ion!like transition state and a proposed glucosyl!enzyme intermediate from an axial direction[

H A

OH HO HO

O

O

OH

R

O

O+ OH O

O–

O

H

OH HO HO

A R

O–

–

A

OH HO HO

O O

H A

OH HO HO

O

H

OH O

OH HO HO

OH

O H

O+

H O

A H

OH O

OH O

O–

O–

Scheme 3

The cyanogenic b!glucosidases from white clover and cassava have the closest homology to the Family 0 b!glucosidase from A`robacterium spp[80 and in this protein Glu!247 has been identi_ed as the catalytic nucleophile[81 This glutamate lies within the highly conserved peptide I:VTENG\ which is also present in the two cyanogenic b!glucosidases[ The acid catalyst group "Glu!087# was identi_ed in the cassava cyanogenic b!glucosidase by a.nity labeling with the inhibitor\ N!bromoacetyl!b!D!glucosylamine[80 This amino acid "Glu!087# also lies within a highly conserved peptide "NEP#[ The crystal structure of the white clover cyanogenic b!glucosidase has been solved at 1[04 _ resolution[8 The overall fold of the molecule is an "a:b#7 barrel\ a structure found in a number of other glycosyl hydrolases\ with all of the residues located in a single domain[ Residues Glu!072 "in NEP# and Glu!286 "in I:VTENG# are highly conserved and are predicted to be the acid catalyst "proton donor# and the nucleophile catalyst "for stabilization of the glycosylium cation!like transition state#\ respectively[ These roles are consistent with the molecular environments of these two residues in the white clover enzyme[ The pocket itself is typical of a sugar!binding site as it contains a number of charged\ aromatic and polar groups[ Molecular modeling has shown that the active site of the protein encoded by the cassava cyanogenic b!glucosidase cDNA has high structural homology to the white clover protein[82

a!hydroxynitrile lyase "acetone cyanohydrin lyase#d a!hydroxynitrile lyase "acetone cyanohydrin lyase#d a!hydroxynitrile lyase "acetone cyanohydrin lyase#

linamarinc

valine

valine

valine

Cassava "Manihot esculenta#

Flax "Linum usitatissimum#

Rubber "Hevea brasiliensis#

c

"S#!aliphatic

"R#!aliphatic

"S#!aliphatic

87

091

32

86

090

NSe "S#!aromatic

099

85

Ref[

"R#!aromatic

"R#!aromatic

Catalyzed synthesis of cyanohydrins

The same biosynthetic enzymes produce linamarin FAD\ ~avin prosthetic group^ CHO\ oligosaccharide^ e

59 999 MW\ monomer\ FAD\e PMSFe inhib[\ glycoprotein 19 999 MW\ homomultimer\ no FAD\ no PMSF inhib[\ no CHOe 27 999 MW\ monomer\ no FAD\ PMSF inhib[ NS\ glycoprotein 22 999 MW¦07 999 MW hetero! tetramer\ no FAD\ DFPe inhib[\ glycoprotein 18 999 MW\ homotrimer\ no FAD\ PMSF inhib[\ no CHO 31 999 MW\ dimer\ no FAD\ PMSF inhib[ NS\ no CHO 29 999 MW\ no FAD\ no CHO

Properties of a!hydroxynitrile lyase

Seeds of Prunus species accumulate the diglucoside "R#!amygdalin\ which is produced by further glycosylation of "R#!prunasin[ b Diglycoside "glucose!arabinose#[ from valine and "R#!lotaustralin from isoleucine[ d There is no evidence for a separate acetone cyanohydrin and "R#!butanone cyanohydrin lyase in cassava or in ~ax[ PMSF\ phenylmethanesulfonyl ~uoride^ DFP\ diisopropyl~uorophosphate^ NS\ not studied[

a

linamarinc

linamarinc

"S#!p!hydroxymandelonitrile lyase

"S#!dhurrin

tyrosine

Sor`hum bicolor

"S#!mandelonitrile lyase

"S#!sambunigrin

phenylalanine

Ximenia americana

"R#!mandelonitrile lyase

Phlebodium aureum "fern#

"R#!vicianinb

phenylalanine

Black cherry "Prunus serotina#

phenylalanine

a!Hydroxynitrile lyase "R#!mandelonitrile lyase

Cyano`lucosides "R#!prunasina

Plant

Precursor amino acids

Table 0 Major plant cyanoglucosides and associated a!hydroxynitrile lyases[

781 Biosynthesis and De`radation of Cyano`enic Glycosides

Biosynthesis and De`radation of Cyano`enic Glycosides

782

The cyanogenic b!glucosidase is encoded by a multigene family in cassava[ All of the genes contain 01 introns and the sequence variation between them re~ects conservation of those amino acid residues which are important in the structure and function of the protein[82 The promoter of one of these cassava genes has been analyzed using reporter gene expression\82 and this analysis indicates that the gene encodes a root!speci_c cyanogenic b!glucosidase[ In white clover\ which is polymorphic for cyanogenesis\ the locus Li controls the presence of cyanogenic b!glucosidase activity in plants\ such that plants homozygous for the nonfunctional allele\ ac\ have no enzyme activity[06 These plants have been shown to produce no cyanogenic b!glucosidase transcript "mRNA#[83 These observations suggest that there is only one gene encoding the cyanogenic b!glucosidase in white clover[

0[20[3[1 a!Hydroxynitrile Lyases A number of a!hydroxynitrile lyases from cyanogenic plant species have been characterized\ largely because there is considerable interest in the use of a!hydroxynitrile lyases as biocatalysts for the synthesis of optically active a!hydroxynitriles\ which are important building blocks in the _ne chemical and pharmaceutical industries[84 Table 0 summarizes the known properties of a!hydroxynitrile lyase enzymes from seven plant species[ From this list the genes for a!hydroxynitrile lyase from black cherry\85 sorghum\86 cassava\32 and Hevea87 have been cloned as cDNA[ It is clear from Table 0 and comparison of the cDNA deduced amino acid sequences that\ unlike the b!glucosidases\ there is little structural similarity among the a!hydroxynitrile lyases from di}erent taxonomic groups[ The only two enzymes which show sequence homology are those from cassava and Hevea\ which are both members of the Euphorbiaceae[ This structural heterogeneity is con_rmed by serological cross!reactivity of hydroxy! nitrile lyases[ Thus\ for example\ antibodies raised against the cassava enzyme\ cross!react with the Hevea proteins but not with the other enzymes\ including the a!hydroxynitrile lyase from ~ax "Linaceae#\ which has the same substrate[88 These enzymes are therefore a good example of con! vergent evolution[ Although the deduced amino acid sequences of the cloned Prunus enzyme85 and the cassava: Hevea32\87 enzyme show no signi_cant homology with other proteins in the databanks\ the a! hydroxynitrile lyase from sorghum\86 shows high sequence homology with a wheat serine car! boxypeptidase[ In addition\ the sorghum enzyme contains the carboxypeptidase catalytic triad\ Ser\ Asp\ His and is inhibited by serine:cysteine modifying agents[ Despite the lack of sequence homology\ cassava a!hydroxynitrile lyase also contains this catalytic triad and is similarly inhibited by the serine protease inhibitor\ phenylmethanesulfonyl ~uoride "PMSF#[092 Site!directed muta! genesis of Ser!79\ which is part of a typical serine protease G!X!S!X!G:A consensus motif\ in this cassava enzyme con_rms that it is essential for enzyme activity[093 The crystal structure of the H[ brasiliensis Muell[ Arg\ a!hydroxynitrile lyase has been determined at 0[8 _ resolution[7 It belongs to the a:b hydrolase superfamily\ with an active site containing the catalytic triad Ser!79\ Asp!196\ His!196 deeply buried within the protein and connected to the surface by a narrow tunnel[ By analogy to other a:b hydrolases\ the reaction catalyzed by the Hevea and cassava a!hydroxynitrile lyase involves a tetrahedral hemiketal or hemiacetal intermediate\ formed by nucleophilic attack of Ser!79 on the substrate\ stabilized by the oxyanion hole[

0[20[4 REFERENCES 0[ A[ Nahrstedt\ in {{Cyanide Compounds in Biology\|| CIBA Found[ Symp[ 039\ eds[ D[ Evered and S[ Harnett\ Wiley\ Chichester\ 0877\ p[ 0201[ 1[ G[ Peiser\ T[!T[ Wang\ N[ E[ Ho}man\ S[ F[ Yang\ H[!W[ Lui\ and C[ T[ Walsh\ Proc[ Natl[ Acad[ Sci[ USA\ 0873\ 70\ 2948[ 2[ E[ E[ Conn\ in {{The Biochemistry of Plants[ A Comprehensive Treatise] Secondary Plant Products\|| eds[ P[ K[ Stumpf and E[ E[ Conn\ Academic Press\ New York\ 0870\ vol[ 6\ p[ 368[ 3[ D[ S[ Seigler\ in {{Herbivores] Their Interactions with Secondary Plant Metabolites*the Chemical Participants\|| eds[ G[ A[ Rosenthal and M[ R[ Berenbaum\ Academic Press\ New York\ 0880\ vol[ 0\ p[ 24[ 4[ J[ E[ Poulton\ Plant Physiol[\ 0889\ 83\ 390[ 5[ O[ Sibbesen\ B[ M[ Koch\ B[ A[ Halkier\ and B[ L[ Mo ller\ Proc[ Natl[ Acad[ Sci[ USA\ 0883\ 80\ 8639[ 6[ O[ Sibbesen\ B[ M[ Koch\ B[ A[ Halkier\ and B[ L[ Mo ller\ J[ Biol[ Chem[\ 0884\ 169\ 2495[ 7[ U[ G[ Wagner\ M[ Hasslacher\ H[ Griengl\ H[ Schwab\ and C[ Kratky\ Structure\ 0885\ 3\ 700[ 8[ T[ Barrett\ C[ G[ Suresh\ S[ P[ Tolley\ E[ J[ Dodson\ and M[ A[ Hughes\ Structure\ 0884\ 2\ 840[ 09[ E[ E[ Conn\ Annu[ Rev[ Plant Physiol[\ 0879\ 20\ 322[

783

Biosynthesis and De`radation of Cyano`enic Glycosides

00[ D[ S[ Seigler\ in {{Progress in Phytochemistry\|| eds[ L[ Reinhold\ J[ B[ Harborne\ and T[ Swain\ Springer Verlag\ Berlin\ 0866\ vol[ 3\ p[ 72[ 01[ P[ A[ Lizotte and J[ E[ Poulton\ Plant Physiol[\ 0877\ 75\ 211[ 02[ H[ Pourmohseni\ W[ D[ Ibenthal\ R[ Machinek\ G[ Remberg\ and V[ Wray\ Phytochemistry\ 0882\ 22\ 184[ 03[ E[ Swain\ C[!P[ Li\ and J[ E[ Poulton\ Plant Physiol[\ 0881\ 87\ 0312[ 04[ C[ R[ Smith\ D[ Weisleder\ and R[ W[ Miller\ J[ Or`[ Chem[\ 0879\ 34\ 496[ 05[ S[ G[ Saupe\ in {{Phytochemistry and Angiosperm Phylogeny\|| eds[ D[ A[ Young and D[ S[ Seigler\ Praeger\ New York\ 0870\ p[ 79[ 06[ M[ A[ Hughes\ Heredity\ 0880\ 55\ 094[ 07[ D[ A[ Jones\ in{{Cyanide Compounds in Biology\|| CIBA Found[ Symp[ 039\ eds[ D[ Evered and S[ Harnett\ Wiley\ Chichester\ 0877\ p[ 040[ 08[ H[ Daday\ Heredity\ 0843\ 7\ 50[ 19[ H[ Daday\ Heredity\ 0843\ 7\ 266[ 10[ G[ A[ Pederson\ T[ E[ Fairbrother\ and S[ L[ Greene\ Crop Sci[\ 0885\ 25\ 316[ 11[ P[ J[ Schappert and J[ S[ Shore\ Heredity\ 0884\ 63\ 281[ 12[ D[ B[ Collinge and M[ A[ Hughes\ J[ Exp[ Bot[\ 0871\ 22\ 043[ 13[ P[ N[ Okolie and B[ N[ Obasi\ Phytochemistry\ 0882\ 22\ 664[ 14[ D[ Selmar\ R[ Lieberei\ N[ Junqueira\ and B[ Biehl\ Phytochemistry\ 0880\ 29\ 1024[ 15[ I[ Shreiner\ D[ Nafus\ and D[ Pimentel\ Ecol[ Entomol[\ 0873\ 8\ 58[ 16[ A[ C[ Bellotti and L[ Riis\ in {{Acta Horticulturae Number 264] International Workshop on Cassava Safety\|| eds[ M[ Bokanga\ A[ J[ A[ Essers\ N[ Poulter\ H[ Rosling\ and O[ Tewe\ WOCAS:ISIS:ISATRC\ Wageningen\ Netherlands\ 0883\ p[ 030[ 17[ D[ Raubenheimer\ M[Sc[ Thesis\ University of Capetown\ 0876[ 18[ G[ Holzkamp and A[ Nahrstedt\ Insect Biochem[ Mol[ Biol[\ 0883\ 13\ 050[ 29[ K[ Honda\ W[ Nishii\ and N[ Hayashi\ J[ Chem[ Ecol[\ 0886\ 12\ 212[ 20[ J[ Lehman\ E[ Meister\ A[ Gutzwiller\ F[ Jans\ J[ P[ Charles\ and J[ Blum\ Rev[ Suisse A`ric[\ 0880\ 12\ 096[ 21[ R[ Lieberei\ R[ Fock\ and B[ Biehl\ An`ew[ Bot[\ 0885\ 69\ 129[ 22[ R[ Lieberei\ J[ Phytopathol[\ 0877\ 011\ 43[ 23[ D[ Selmar\ R[ Lieberei\ and B[ Biehl\ Plant Physiol[\ 0877\ 75\ 600[ 24[ C[ R[ Smith Jr\ D[ Weisleder\ R[ W[ Miller\ I[ S[ Palmer\ and O[ E[ Olsen\ J[ Or`[ Chem[\ 0879\ 34\ 496[ 25[ D[ Selmar\ Z[ Irandoost\ and V[ Wray\ Phytochemistry\ 0885\ 32\ 458[ 26[ S[ R[ A[ Adewusi\ Plant Physiol[\ 0889\ 83\ 0108[ 27[ M[ Kojima\ J[ E[ Poulton\ S[ S[ Thayer\ and E[ E[ Conn\ Plant Physiol[\ 0868\ 52\ 0911[ 28[ S[ S[ Thayer and E[ E[ Conn\ Plant Physiol[\ 0870\ 56\ 506[ 39[ W[ L[ B[ White\ J[ M[ McMahon\ and R[ T[ Sayre\ in {{Acta Horticulturae Number 264] International Workshop on Cassava Safety\|| eds[ M[ Bokanga\ A[ J[ A[ Essers\ N[ Poulter\ H[ Rosling\ and O[ Tewe\ WOCAS:ISIS:ISATRC\ Wageningen\ Netherlands\ 0883\ p[ 58[ 30[ A[ Pancoro and M[ A[ Hughes\ Plant J[\ 0881\ 1\ 710[ 31[ M[ Elias\ B[ Nambisan\ and P[ R[ Sudhakaran\ Arch[ Biochem[ Biophys[\ 0886\ 230\ 111[ 32[ J[ Hughes\ F[ J[ P[ De C[ Carvalho\ and M[ A[ Hughes\ Arch[ Biochem[ Biophys[\ 0883\ 200\ 385[ 33[ P[ Kakes\ Planta\ 0874\ 055\ 045[ 34[ M[ Frehner and E[ E[ Conn\ Plant Physiol[\ 0876\ 73\ 0185[ 35[ C[ Gruhnert\ B[ Biehl\ and D[ Selmar\ Planta\ 0883\ 084\ 25[ 36[ H[!C[ Wu and J[ E[ Poulton\ Plant Physiol[\ 0880\ 85\ 0218[ 37[ E[ Swain\ C[ P[ Li\ and J[ E[ Poulton\ Plant Physiol[\ 0881\ 87\ 0312[ 38[ E[ Swain and J[ E[ Poulton\ Plant Physiol[\ 0883\ 095\ 326[ 49[ J[ E[ Poulton and C[ P[ Li\ Plant Physiol[\ 0883\ 093\ 18[ 40[ L[ Zheng and J[ E[ Poulton\ Plant Physiol[\ 0884\ 098\ 20[ 41[ B[ R[ Maslin\ E[ E[ Conn\ and J[ E[ Dunn\ Phytochemistry\ 0874\ 13\ 850[ 42[ J[ H[ Cock\ in {{Cassava] New Potential for a Neglected Crop\|| West_eld Press\ London\ 0874\ p[ 080[ 43[ D[ B[ Collinge and M[ A[ Hughes\ Plant Sci[ Lett[\ 0873\ 23\ 008[ 44[ J[ Lykkesfeldt and B[ L[ Mo ller\ Acta Chem[ Scand[\ 0883\ 37\ 067[ 45[ R[ Cook\ N[ McCaig\ J[ M[ B[ McMillan\ and W[ B[ Lumsden\ J[ Inst[ Brew[\ 0889\ 85\ 122[ 46[ A[ Nahrstedt\ in {{Biologically Active Natural Products\|| eds[ K[ Hostettmann and P[ J[ Lea\ Clarendon Press\ Oxford\ 0876\ p[ 102[ 47[ J[ Koukol\ P[ Miljanich\ and E[ E[ Conn\ J[ Biol[ Chem[\ 0851\ 126\ 2112[ 48[ I[ J[ McFarlane\ E[ M[ Lees\ and E[ E[ Conn\ J[ Biol[ Chem[\ 0864\ 149\ 3697[ 59[ M[ Shimada and E[ E[ Conn\ Arch[ Biochem[ Biophys[\ 0866\ 079\ 088[ 50[ B[ L[ Mo ller and E[ E[ Conn\ J[ Biol[ Chem[\ 0868\ 143\ 7464[ 51[ B[ A[ Halkier\ C[ E[ Olsen\ and B[ L[ Mo ller\ J[ Biol[ Chem[\ 0878\ 153\ 08 376[ 52[ B[ A[ Halkier and B[ L[ Mo ller\ J[ Biol[ Chem[\ 0889\ 154\ 10 003[ 53[ B[ A[ Halkier\ J[ Lykkesfeldt\ and B[ L[ Mo ller\ Proc[ Natl Acad[ Sci[ USA\ 0880\ 77\ 376[ 54[ B[ A[ Halkier and B[ L[ Mo ller\ Plant Physiol[\ 0880\ 85\ 09[ 55[ B[ A[ Halkier\ H[ L[ Nielsen\ B[ M[ Koch\ and B[ L[ Mo ller\ Arch[ Biochem[ Biophys[\ 0884\ 211\ 258[ 56[ B[ M[ Koch\ O[ Sibbesen\ B[ A[\ Halkier\ I[ Svendsen\ and B[ L[ Mo ller\ Arch[ Biochem[ Biophys[\ 0884\ 212\ 066[ 57[ D[ B[ Collinge and M[ A[ Hughes\ Arch[ Biochem[ Biophys[\ 0871\ 107\ 27[ 58[ A[ J[ Cutler and E[ E[ Conn\ Arch[ Biochem[ Biophys[\ 0870\ 101\ 357[ 69[ B[ M[ Koch\ V[ S[ Nielsen\ B[ A[ Halkier\ C[ E[ Olsen\ and B[ L[ Mo ller\ Arch[ Biochem[ Biophys[\ 0881\ 181\ 030[ 60[ D[ B[ Collinge and M[ A[ Hughes\ Plant Sci[ Lett[\ 0873\ 23\ 008[ 61[ M[ A[ Hughes and E[ E[ Conn\ Phytochemistry\ 0865\ 04\ 576[ 62[ W[ Hosel and A[ Nahrstedt\ Arch[ Biochem[ Biophys[\ 0879\ 192\ 642[ 63[ J[ E[ Poulton and S[!I[ Shin\ Z[ Naturforsch[\ 0872\ 27c\ 258[ 64[ W[ Hosel and O[ Schiel\ Arch[ Biochem[ Biophys[\ 0873\ 118\ 066[

Biosynthesis and De`radation of Cyano`enic Glycosides 65[ 66[ 67[ 68[ 79[ 70[ 71[ 72[ 73[ 74[ 75[ 76[ 77[ 78[ 89[ 80[ 81[ 82[ 83[ 84[ 85[ 86[ 87[ 88[ 099[ 090[ 091[ 092[ 093[

784

K[ Hahlbrock and E[ E[ Conn\ J[ Biol[ Chem[\ 0869\ 134\ 806[ P[ F[ Reay and E[ E[ Conn\ J[ Biol[ Chem[\ 0863\ 138\ 4715[ H[ Mederacke\ D[ Selmar\ and B[ Biehl\ An`ew[ Bot[\ 0884\ 58\ 008[ G[ Hrazdina and G[ J[ Wagner\ Annu[ Proc[ Phytochem[ Soc[ Europe\ 0874\ 14\ 019[ J[ Hughes and M[ A[ Hughes\ DNA Sequence\ 0883\ 4\ 30[ E[ E[ Conn\ in {{b!Glucosidases] Biochemistry and Molecular Biology\|| ed[ A[ Esen\ ACS Symposium Series 422\ American Chemical Society\ Washington DC\ 0882\ p[ 04[ T[ W[!M[ Fan and E[ E[ Conn\ Arch[ Biochem[ Biophys[\ 0874\ 132\ 250[ D[ Selmar\ Ph[D[ Thesis\ University of Braunschweig\ 0875[ T[ Kasai\ M[ Kisimoto\ and S[ Kawamura\ Ka`awa Dia`aku Na`akubu Hokoku\ 0870\ 21\ 000[ C[ P[ Li\ E[ Swain\ and J[ E[ Poulton\ Plant Physiol[\ 0881\ 099\ 171[ E[ Oxtoby\ M[ A[ Dunn\ A[ Pancoro\ and M[ A[ Hughes\ Plant Mol[ Biol[\ 0880\ 06\ 198[ M[ A[ Hughes\ K[ Brown\ A[ Pancoro\ B[ S[ Murray\ E[ Oxtoby\ and J[ Hughes\ Arch[ Biochem[ Biophys[\ 0881\ 184\ 162[ B[ Henrissat\ Biochem[ J[\ 0880\ 179\ 298[ A[ Rojas\ L[ Arola\ and A[ Romeu\ Biochem[ Mol[ Biol[ Int[\ 0884\ 24\ 0112[ D[ Trimbur\ R[ A[ J[ Warren\ and S[ G[ Withers\ in {{b!Glucosidases] Biochemistry and Molecular Biology\|| ed[ A[ Esen\ ACS Symposium Series 422\ American Chemical Society\ Washington DC\ 0882\ p[ 31[ Z[ Kerseztessy\ L[ Kiss\ and M[ A[ Hughes\ Arch[ Biochem[ Biophys[\ 0883\ 204\ 212[ S[ G[ Withers\ R[ A[ J[ Warren\ I[ P[ Street\ K[ Rupitz\ J[ B[ Kempton\ and R[ Aebersold\ J[ Am[ Chem[ Soc[\ 0889\ 001\ 4776[ S[ Liddle\ Z[ Keresztessy\ J[ Hughes\ and M[ A[ Hughes\ Afr[ J[ Root Tuber Crops\ 0886\ 1\ 047[ M[ A[ Hughes and M[ A[ Dunn\ Plant Mol[ Biol[\ 0871\ 0\ 058[ H[ Wajant and F[ E}enberger\ Biol[ Chem[\ 0885\ 266\ 500[ I[!P[ Cheng and J[ E[ Poulton\ Plant Cell Physiol[\ 0882\ 23\ 0028[ H[ Wajant\ K[!W[ Mundry\ and K[ P_zenmaier\ Plant Mol[ Biol[\ 0883\ 15\ 624[ M[ Hasslaeher\ M[ Schall\ M[ Hayn\ H[ Griengl\ S[ D[ Kohlwein\ and H[ Schwab\ J[ Biol[ Chem[\ 0885\ 160\ 4773[ H[ Wajant\ S[ Forster\ H[ Bottinger\ F[ E}enberger\ and K[ P_zenmaier\ Plant Sci[\ 0884\ 097\ 0[ H[ Wajant\ S[ Forster\ D[ Selmar\ F[ E}enberger\ and K[ P_zenmaier\ Plant Physiol[\ 0884\ 098\ 0120[ G[ W[ Kuroki and E[ E[ Conn\ Proc[ Natl Acad[ Sci[ USA\ 0875\ 75\ 5867[ L[!L[ Xu\ B[ K[ Singh\ and E[ E[ Conn\ Arch[ Biochem[ Biophys[\ 0877\ 155\ 145[ J[ Hughes\ J[ H[ Lakey\ and M[ A[ Hughes\ Biotechnol[ Bioen`[\ 0886\ 42\ 221[ H[ Wajant and K[ P_zenmaier\ J[ Biol[ Chem[\ 0885\ 160\ 14 729[