Sulfur Biochemistry of Garlic: the Biosynthesis of Flavor Precursors

H.A. Collin, J. Hughes, A. Tregova, J.G.C. Milne, G. van der Werff, M. Wilkinson, M.G. Jones and A.B. Tomsett The School of Biological Sciences, The Biosciences Building, Crown Street, The University of Liverpool, Liverpool L69 7ZB United Kingdom

R. Cosstick The Department of Chemistry, Robert Robinson Laboratories, The University of Liverpool, Liverpool L69 7ZD, United Kingdom

L. Trueman, T. Crowther, L. Brown and B Thomas HRI Wellesbourne, Wellesbourne, Warwick, CV35 9EF, United Kingdom

Keywords: Allium sativum, alliin, cysteine synthase, sulfur metabolism, -glutamyl cysteine sulfoxide

Abstract The major flavour precursors in the genus Allium include alliin and isoalliin, members of the alk(en)yl cysteine sulfoxide group (CSOs). There are also large amounts of S-alk(en)yl gamma-glutamyl-cysteine sulfoxide compounds. It has been suggested that alliin is derived from serine and an allyl thiol source, or from glutathione and an allyl source, while isoalliin is synthesised via glutathione and a series of gamma-glutamyl peptides. We have combined several approaches to investigate the intermediates and enzymes involved in the biosynthetic pathways. These include profiling and tracking sulfur components in garlic leaf, clove and root tissue as bulbs develop in the greenhouse. This has allowed us to build up, for the first time, a picture of the dynamic uptake, allocation and distribution of flavour precursors during the life cycle. This indicated that CSOs are remobilised to the new cloves in a late stage of development, following uptake of the bulk of sulfur by the plant. Experiments involving feeding potential intermediates to onion and garlic callus tissue, that generally lack flavour precursors, indicated that several alk(en)yl thiols could be converted to alk(en)yl cysteine and alk(en)yl cysteine sulfoxide by callus. This may indicate that synthase and oxidase enzyme(s) with broad substrate specificity may exist in Allium. Using data derived from the scientific literature, we have searched for genes and enzymes that may be involved in the biosynthetic pathway. Cysteine synthase (CS) and serine acetyltransferase (SAT) may play a role in flavour precursor biosynthesis. Sequences with significant homology to two groups of plant CS and to a SAT have been obtained from garlic (cultivar Printanor). The sequence of one further CS has been identified, using partial peptide sequences from a protein that showed allyl cysteine synthase activity. Our results provide further insight into the biosynthetic pathway of garlic flavour compounds.

INTRODUCTION Garlic (Allium sativum L.) has long been valued for adding flavour to food and health benefits are also attributed to it (Kik et al., 2001). Both originate in damaged tissues from enzymatic cleavage of pre-formed stable flavour precursors by alliinase. The flavour precursors are secondary metabolites, the alk(en)yl cysteine sulfoxides (CSOs), primarily allyl cysteine sulfoxide (ACSO, alliin, 2-propenyl cysteine sulfoxide) and methyl cysteine sulfoxide (MCSO, methiin). The synthesis and storage of these compounds, and related -glutamyl cysteine sulfoxide peptides (-GP), results in garlic containing the highest amount of sulfur recorded in any plant species (Nielsen et al., 1991). Garlic sulfur compounds have shown potential in combating cardiovascular disease and some cancers (Kik et al., 2001), and there is therefore interest in improving the content of these compounds in garlic. However, understanding of the biosynthetic pathway is minimal, especially when compared with current knowledge of the assimilation of sulfur from sulfate to cysteine (Leustek, 2000). Two routes have been proposed, one via glutathione (Lancaster and Shaw, 1989) and one via serine (Granroth, 1970) as illustrated in Fig. 1. For both pathways, some intermediates and the majority of genes and enzymes remain to be identified. It is possible that each pathway dominates at a different time in the life-cycle or in different organs. During its biennial life-cycle the bulb of garlic cloves develops from axillary buds during late summer. These act as over-wintering storage organs, with re-use of stored carbon, nitrogen and sulfur resources from CSOs and -GPs as roots and leaves develop at the start of the growing season. During active growth, fresh sulfur is assimilated from the soil via the roots. There is evidence that CSO synthesis occurs in the leaf, with products being translocated to the developing bulb towards the end of the growing season (Lancaster et al., 1986). The cloves also contain substantial amounts of -GPs. The content and relationship of CSOs and -GPs are thus the result of biosynthesis and resource allocation. A greater understanding of sulfur partition during the life cycle and the biosynthetic pathways for CSOs and -GPs would aid development of varieties and agronomic procedures to yield high quality garlic with an improved content of flavour precursors. To this end, we have reviewed information in the scientific literature for CSO synthesis in Allium and other species and initiated research, focusing on the role of cysteine synthases.

CSO SYNTHESIS IN ALLIUMS Experimental studies on flavour precursor biosynthesis in onion (Allium cepa L.) and garlic leaves, roots and tissue cultures, summarised in Table 1, shows that the addition of exogenous thiols or alk(en)yl cysteines can give rise to the corresponding alk(en)yl cysteines or alk(en)yl cysteine sulfoxides. In several studies, radiolabelled precursors were used and indicated quantitative conversion to radiolabelled products (Parry and Sood, 1989; Ohsumi et al., 1993). In addition, these studies indicated that onion roots and both onion and garlic callus could synthesise a wide range of alk(en)yl cysteines and alk(en)yl cysteine sulfoxides when provided with suitable precursors. For example, provision of benzenemethane thiol and ethane thiol result in synthesis of benzyl cysteine and ethyl cysteine, respectively (Granroth, 1970; Prince et al., 1997). The former has not been detected within Allium sp. while the latter has only been occasionally reported in trace amounts (Kubec et al., 2000). Flavour precursors are normally absent from callus, or present at only trace levels, indicating that biosynthetic capacity is not complete but the studies summarised in Table 1 indicate that synthesis is possible with provision of suitable substrates. An advantage of callus is that exogenous substrates can enter the intact tissue through the thin cuticle that develops in the humid culture conditions. This avoids any problems with cleavage of flavour precursors by alliinase that might occur in damaged tissue. This experimental information therefore, although not excluding the glutathione pathway, supports the serine biosynthetic route as illustrated in Fig. 1B.

CSO SYNTHESIS IN OTHER PLANT SPECIES Synthesis of MCSO is not confined to Allium sp., but also occurs in the Brassicaceae. Additionally, other CSOs have been reported in several unrelated tropical plants and within fungi. For example, roots of the perennial Amazonian shrub garlic weed (Petiveria alliacea, Phytolaccaceae) contain benzyl cysteine sulfoxide and several species of the basidiomycete Marasmius contain methylthiomethyl cysteine-4-oxide (marasmine) (see Jones et al., 2004 for a summary of these reports). The implication is that this secondary metabolic capacity may have evolved on more than one occasion, and that Allium sp. are only one, particularly intensively studied, example. Secondary products are frequently synthesised by pathways with parallels in primary metabolism, following gene duplication and mutation. Reports of CSO synthesis by primary metabolic enzymes are thus particularly interesting. Ikegami and Murakoshi (1994) reviewed the synthesis of several non-protein -substituted alanines and some higher homologues in plants, indicating that cysteine synthase is the key biosynthetic enzyme. Alk(en)yl cysteines are members of this class of compound and could be synthesised, in vitro, by cysteine synthase isolated from a surprisingly wide range of plants. Before considering the significance of this finding for flavour precursor biosynthesis in garlic, a description of the role of cysteine synthase in primary cell metabolism is necessary.

CYSTEINE SYNTHESIS The synthesis of the aminoacid cysteine is catalysed by two consecutive enzymes, serine acetyltransferase (SAT, EC 2.3.1.30) and cysteine synthase (CS, also known as O-acetylserine thiol lyase (EC 4.2.99.8)) (see Fig. 2.). The former activates serine to the intermediary compound O-acetyl serine (OAS), while the latter catalyses the formation of cysteine through insertion of sulfide into O-acetyl serine (Saito, 2000). The activity of SAT is the rate-limiting step in cysteine synthesis in planta. This enzyme is catalytically active when in a heteromeric complex with CS, although the OAS synthesised in the complex is released to be utilised by free CS for cysteine synthesis. The overall reaction is a key step in sulfur assimilation in plants, since cysteine is the first organic sulfur compound formed within the cell, uniting carbon, nitrogen and sulfur metabolism. The process is tightly regulated at both transcriptional and post- translational levels, the latter operating through protein-protein interactions, sub-cellular compartmentation and modulation by substrates and products (Saito et al., 2000). At least three isoforms of CS and SAT have been identified in plants, located in the cytosol, chloroplast and mitochondrion, although the need for cysteine synthesis in each compartment is not clear. CS contains a pyridoxal 5’-phosphate co-factor and has now been classified within the subfamily of -substituted alanine synthase in the large superfamily of pyridoxal-phosphate enzymes (Hatzfeld et al., 2000) Despite the obvious importance of cysteine synthesis in plant primary metabolism, there is substantial evidence that some CSs have evolved additional biochemical properties. Activity as a cyanoalanine synthase has been attributed to the mitochondrial CS isoforms from spinach (Spinacia oleracea L.) and Arabidopsis thaliana (Hatzfeld et al., 2000). Mitochondria in some plants can detoxify cyanide, produced during ethylene biosynthesis, through synthesis of cyanoalanine from cysteine and cyanide, liberating sulfide. Although recombinant cytosolic and chloroplastic forms of spinach CS could also synthesise cyanoalanine, they displayed low cyanoalanine synthase activity relative to the rates of cysteine synthesis. This contrasted with the mitochondrial isoform, which was more active in cyanoalanine than cysteine synthesis. Measurement of Km values showed that the mitochondrial isoforms had a much higher affinity for cyanide than sulfide, and the reverse was true for the cytosolic and chloroplastic forms (Hatzfeld et al., 2000). Although the relative ratio of these two reactions in vivo is likely to be controlled by the availability of sulfide and cyanide, structural changes in the enzymes may also affect substrate usage. Further kinetic and protein sequence analysis showed that a single aminoacid substitution in the substrate-binding pocket of the enzyme may hinder OAS binding (Warrilow and Hawkesford, 2002) and that the major mechanistic difference between CS and cyanoalanine synthase probably lies in the binding and processing of the sulfide or cyanide anions.

CYSTEINE SYNTHASES IN SECONDARY METABOLISM There is substantial evidence that some CSs are active in secondary metabolism. Early studies on cysteine synthesis in plants indicated that thiols, as well as sulphide, were substrates in vitro (e.g. Thompson and Moore, 1968). Heterocyclic substituted alanines are present in several plants and are of interest because of their pharmacological and toxic properties. Examples include mimosine from members of the Leguminosae which has thyrotoxic and depilatory properties and -(isoxazolin-5-on-2-yl)-alanine from Pisum sp. and Lathyrus sp. which has antifungal activity (Ikegami and Murakoshi, 1994). These are synthesised by condensation of an appropriate N-heterocyclic compound with OAS. Initial studies on the biosynthetic enzyme from two plants indicated that both required OAS but had different specificities for the heterocyclic acceptor. Tests of a range of acceptors showed that both enzymes could catalyse the synthesis of methyl cysteine in addition to the natural product. Purification of several enzymes for -substituted alanine synthesis indicated that several of them co-purified with peaks in CS activity (Ikegami et al., 1990). This circumstantial evidence for the identity of the -substituted alanine biosynthetic enzymes as a type of CS was strengthened by studies of the physicochemical and kinetic properties of two isoenzymes from eight species that had been purified to apparent homogeneity. The species included model plants such as spinach (Spinacia oleracea) and pea (Pisum sativum) as well as species that produced unusual -substituted alanines such as rangoon creeper (Quisqualis indica, Combretaceae). The physical properties of the enzymes were within the range typical of CS (Ikegami and Murakoshi, 1994). In a standard assay system, OAS was the sole alanyl donor and each enzyme demonstrated a distinct substrate specificity against a panel of N-heterocyclic and thiol compounds, relative to synthesis of cysteine. All could form methyl cysteine, allyl cysteine and carboxymethyl cysteine from OAS and the appropriate thiol, but at very different rates. In most, rates were only a few per cent of that for cysteine synthesis. Interestingly, CS isoenzyme B from the leadtree (Leucaena leucocephala, Fabaceae), a tree forage legume native to tropical America, could synthesis allyl cysteine at a rate 73.2% of that for cysteine, while isoenzyme A could synthesise methyl cysteine and allyl cysteine at 29.6% and 21.6% respectively. Isoenzyme B from watermelon (Citrullus vulgaris, Cucurbitaceae) was also able to synthesise methyl cysteine at 71.9% that of cysteine. The natural -substituted alanines synthesised in these two plants are the N-heterocyclic compounds mimosine and -(pyrazol –1-yl) alanine. Only CS from leadtree and one other of the eight plants could synthesise mimosine, although the rate for even the most effective isoenzyme B from leadtree was only 15.7% that for cysteine. Although isoenzymes from all eight plants could synthesise -(pyrazol –1-yl) alanine, the rates were generally less than 2% of the rate of cysteine synthesis, including the watermelon CSs (Ikegami et al., 1990; Ikegami and Murakoshi, 1994). It is therefore clear that CSs from many plants are capable of synthesising alk(en)yl cysteines when provided with a suitable substrate. The relationship between biosynthetic rates in vitro and substrate specificity in vivo is complex, since in these experiments biosynthetic rates with substrates that would not be encountered in vivo were sometimes higher than for possibly natural substrates.

CYSTEINE SYNTHESIS IN ALLIUMS Comparatively little is known about the genes and enzymes involved in cysteine, CSO or -GP biosynthesis in Allium. The genus may have a greater requirement for cysteine than most plants to support synthesis of the secondary metabolites that may lead to a higher flux and/or intracellular levels. Measurements of total cysteine content in chinese chives (Allium tuberosum) indicated that it was five- to six-fold higher than in Arabidopsis (Urano et al., 2000). Cysteine affects the activity of SAT, which is the rate- limiting step in cysteine synthesis in planta. It has been established that some SATs are inhibited by physiological micromolar levels of cysteine in an allosteric manner (e.g. watermelon, cytosolic Arabidopsis, chloroplastic pea and spinach) while others (e.g. chloroplastic Arabidopsis) are not (Droux, 2003). The cDNA sequences have been obtained for a cytosolic SAT and CS from chinese chive (Urano et al., 2000). Both were expressed constitutively in leaves and roots. Expression of this SAT in Escherichia coli yielded an enzyme activity that was inhibited by cysteine (IC50) at the comparatively high concentration of 48.7 M, about ten-fold higher than typical inhibitory levels. Although tissue levels of cysteine in chinese chives are higher than in some other plants, further work would be required to elucidate whether this difference in IC50 is significant for CSO and -GP synthesis. Two forms of CS have been purified from chinese chive leaves (Ikegami et al., 1993) which displayed similar physical and kinetic properties to CSs from other plants, and were able to synthesise methyl-, allyl- and carboxymethyl- cysteine in vitro when provided with suitable thiol substrates. We have obtained sequences for three distinct CSs from garlic which vary in their tissue expression and ability to synthesise allyl cysteine (Tregova et al., 2004).

PROSPECTS FOR A MOLECULAR UNDERSTANDING OF CSO SYNTHESIS Research into flavour precursor biosynthesis in Allium is at a stage where developments in understanding of cysteine and glutathione metabolism over the last few decades can be applied with the aim of increasing CSO synthesis. We have focused on the role of CS, since a role for this enzyme in the synthesis of -substituted alanines has been demonstrated. It is clear that CSs from many plants are able to synthesise alk(en)yl cysteine when provided with suitable substrates. However, more detailed kinetic studies of recombinant Allium CSs from each cell compartment are needed to provide a better appreciation of their biosynthetic potential. The importance of CS in CSO synthesis in vivo in Allium sp. remains to be determined. Over-expression or silencing of endogenous CS genes might provide an answer, although the effect of transformation of other plant species with genes for SAT and/or CS has had varying results. Some report little effect on phenotype while others have demonstrated plants exhibiting elevated levels of OAS, cysteine, glutathione or other metabolites (Sirko et al., 2004). Since cysteine synthesis is regulated at multiple levels in vivo this variety of effects is not unexpected and careful analysis of transgenic phenotypes would be required. The nature of the genes and enzymes required for sulfoxidation of the S-alk(en)yl group remains to be determined, as well as those required for the proposed glutathione pathway. Their identification is important to obtain the full picture of CSO biosynthesis and to explore the relationships between the glutathione and serine pathways. Additional information on sulphur resource allocation during growth and bulbing would assist in improving agricultural systems for garlic production, and could be used, in conjunction with knowledge about the biosynthetic pathways, to improve garlic varieties through selection among existing cultivars. Although garlic can only be propagated vegetatively at present, if fertile varieties became available these aspects of sulfur biochemistry might provide markers to assist breeders. If transgenic garlic were to be developed as a commercial crop, these studies could determine whether targeting transgenes to particular organelles, tissues or times during development would be beneficial in influencing CSO synthesis.

ACKNOWLEDGEMENTS We would like to thank all our EU Garlic and Health project partners for their comments and advice over the past four years. This research was financed by an EU FP5 grant in the area of Key Action 1 (QLK1-CT-1999-498).

Literature cited Droux M. 2003. Plant serine acetyltransferases: new insights for regulation of sulphur metabolism in plant cells. Plant Physiol. Biochem. 41:619 – 627. Granroth, B. 1970. Biosynthesis and decomposition of cysteine derivatives in onion and other Allium species. Ann. Acad. Sci. Fenn. A 154:1 – 71. Hatzfeld, Y., Maruyama, A., Schmidt, A., Noji, M., Ishizawa, K. and Saito, K. 2000. β- cyanoalanine synthase is a mitochondrial cysteine synthase-like protein in spinach and Arabidopsis. Plant Physiol. 123:1163 – 1171. Hughes, J., Tregova, A., Tomsett, A.B., Jones, M.G, Cosstick, R. and Collin, H.A. 2004. Synthesis of alliin in garlic and onion tissue cultures. Phytochemistry (submitted) Ikegami, F., Mizuno, M., Kihara, M. and Murakoshi, I. 1990. Enzymatic synthesis of the thyrotoxic amino acid mimosine by cysteine synthase. Phytochemistry. 29:3461-3465. Ikegami F., Itagaki S. and Murakoshi I. 1993. Purification and characterization of two forms of cysteine synthase from Allium tuberosum. Phytochemistry 32:31 – 34. Ikegami F. and Murakoshi I. 1994 Enzymic synthesis of non-protein β-substituted alanines and some higher homologues in plants. Phytochemistry 35:1089 – 1104. Jones, M.G., Hughes, J., Tregova, A., Milne, J.C.G., Tomsett, A.B., and Collin, H.A. 2004. Biosynthesis of the flavour precursors of onion and garlic. J. Exp. Bot. (accepted for publication) Kik C., Kahane R. and Gebhardt R., 2001. Garlic and Health. Nutrition, Metabolism and Cardiovascular Diseases 11:57-65 (Suppl) Kubec, R., Svobodova, M., Velíšek J. 2000. Distribution of S-alk(en)ylcysteine sulphoxides in some Allium species. Identification of a new flavor precursor: S- ethylcysteine sulphoxide (ethiin). J. Agri. Food Chem. 48:428 – 433. Lancaster, J.E. and Shaw, M.L. 1989. γ – glutamyl peptides in the biosynthesis of S- alk(en)yl-L-cysteine sulphoxides (flavour precursors) in Allium. Phytochemistry 28:455 – 460. Lancaster, J.E., McCallion, B.J. and Shaw, M.L. 1986. The dynamics of the flavour precursors, the S-alk(en)yl-L-cysteine sulphoxides, during leaf blade and scale development in the onion (Allium cepa). Physiol. Plantarum. 66:293 – 297. Leustek, T., Martin, M.N., Bick, J-A.,and Davies, J.P. 2000. Pathways and regulation of sulfur metabolism revealed through molecular and genetic studies. Ann. Rev. Plant Physiol. and Plant Mol. Biol. 51:141 – 165. Nielsen, K.K., Mahoney, A.W., Williams, L.S. and Rogers, V.C. 1991 X-ray fluorescence measurements of Mg, P, S, Cl, K, Ca, Mn, Fe, Cu and Zn in fruits, vegetables and grain products. J Food Comp Anal. 4:39-51 Ohsumi, C, Hayashi, T. and Sano, K. 1993. Formation of alliin in the culture tissues of allium sativum. Phytochem. 33:107-111 Parry, R.J. and Sood, G.R. 1989. Investigations of the biosynthesis of trans-(+)-S-1- propenyl-L-cysteine sulphoxide in onions (Allium cepa). J. Am. Chem. Soc. 111:4514-4515 Prince, C.L., Shuler, M.L., and Yamada, Y. 1997. Altering flavor profiles in onion (Allium cepa L.) root cultures through directed biosynthesis. Biotechnol. Prog. 13:506 –510. Saito, K. 2000. Regulation of sulfate transport and synthesis of sulfur-containing amino acids. Curr. Op. Plant Biol. 3:188-195 Saito, K., Takahashi, H., Noji, M., Inoue, K., and Hatzfeld, Y. 2000. Molecular regulation of sulfur assimilation and cysteine synthesis. p.59-72. In: C Brunold et al. (eds), Sulfur nutrition and sulfur assimilation in higher plants. Paul Haupt, Bern, Switzerland. Sirko, A., Blaszczyk, A. and Liszewska, F. 2004. Overproduction of SAT and/or OAS- TL in transgenic plants – a survey of effects. Comp. Biochem. Physio. Pt A. 137:S232 Thompson, J.F. and Moore, D.P. 1968. Enzymatic synthesis of cysteine and S- methylcysteine in plant extracts. Biochem. Biophys. Res. Commun. 31:281 – 286 Tregova, A., Hughes, J., Milne, J.G.C., Wilkinson, M., Jones, M.G., Collin, H.A. and Tomsett, A.B. (2004) Novel cysteine synthases from garlic and their role in flavour biosynthesis (manuscript in preparation) Turnbull, A., Galpin, I.J. and Collin, H.A. 1980. Comparison of the onion plant (Allium cepa) and onion tissue culture III. Feeding of 14C labelled precursors of the flavour precursor compounds. New Phytol. 85:483-487 Urano Y., Manabe T., Noji M. and Saito K. 2000. Molecular cloning and functional characterization of cDNAs encoding cysteine synthase and serine acetyltransferase that may be responsible for high cellular cysteine content in Allium tuberosum. Gene 257:269 – 277. Warrilow, A.G.S. and Hawkesford, M.J. 2002. Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by -substituted alanyl and anion inhibitors. J. Exp. Bot. 53:439-445.

Tables

Table 1. The production of substituted cysteines and cysteine sulfoxides in onion and garlic tissues after supplementation with organic thiols or substituted cysteines.

Tissue Substrate Product Reference Onion leaf Methane thiol Methyl cysteine Granroth, 1970 Ethane thiol Ethyl cysteine Propyl thiol Propyl cysteine Allyl thiol Allyl cysteine Benzenemethanethiol Benzyl cysteine 2-Thioethanol 2-Hydroxyethyl cysteine

Methyl cysteine Methyl cysteine sulfoxide Ethyl cysteine Ethyl cysteine sulfoxide Propyl cysteine Propyl cysteine sulfoxide 1-Propenyl cysteine 1-Propenyl cysteine sulfoxide 2-Carboxypropyl 1-Propenyl cysteine sulfoxide cysteine Onion bulb 2-Carboxypropyl 1-Propenyl cysteine sulfoxide Parry and Sood cysteine 1989 Onion callus 2-Carboxypropyl 1-Propenyl cysteine sulfoxide Turnbull et al., cysteine 1980 Onion root Propanethiol Propyl cysteine sulfoxide Prince et al., 1997 cultures 2-Propenethiol Allyl cysteine sulfoxide Ethanethiol Ethyl cysteine sulfoxide

Garlic leaves Methane thiol Methyl cysteine Granroth, 1970 Ethane thiol Ethyl cysteine Propyl thiol Propyl cysteine Allyl thiol Allyl cysteine Differentiating Allyl thiol Allyl cysteine and allyl Ohsumi et al., garlic callus cysteine sulfoxide 1993 Garlic callus Propyl thiol Propyl cysteine and propyl Hughes et al., Propyl cysteine cysteine sulfoxide 2004 Allyl thiol Propyl cysteine sulfoxide Allyl cysteine and allyl cysteine sulfoxide Propenyl cysteine 1-Propenyl cysteine sulfoxide Figures A B

glutathione O-acetyl serine

alk(en)yl donor S-alk(en)yl donor

S- alk(en)yl glutathione S- alk(en)yl cysteine

glycine

γ-glutamyl S- alk(en)yl cysteine

γ-glutamyl S- alk(en)yl cysteine sulphoxide

glutamic acid

S- alk(en)yl cysteine sulphoxide S- alk(en)yl cysteine sulphoxide

Fig. 1. The biosynthetic pathways proposed for Allium flavour precursors. Route A, after Lancaster and Shaw (1989) involves participation of glutathione and γ-glutamyl derivatives. Route B, after Granroth (1970) is consistent with a role for serine (adapted from Jones et al., 2004).

serine acetyltransferase serine + acetylCoA O-acetylserine + CoA

cysteine synthase O-acetylserine + sulfide cysteine + acetate

Fig. 2. The biosynthesis of cysteine (adapted from Leustek et al., 2000)