Introduction

The flavour precursors and their role

Byosynhtesis of flavour precursors

Byosynhtesis of organoselenium compounds

Introduction

This section will focus on current knowledge about the biosynthesis of the flavour precursors, the (+)-S-alk(en)yl cysteine sulphoxides (CSOs) and their γ-glutamyl peptide (γGPs) relatives. Although a biosynthetic pathway has been published, 1 there is still considerable uncertainty about several stages, the relationship between the CSOs and γGPs, and whether the same pathway is followed in all tissues.

Very little is known about some aspects, such as the oxidation step required to form the sulphoxide. There is some information about the subcellular location of flavour biosynthesis, and about the movement of flavour precursors and γGPs during plant development.

The biosynthetic pathways proposed for the Allium flavour precursors are based primarily on chemical analysis and radiotracer studies. Most of the enzyme activities that would be required for the proposed biosynthetic steps are from large protein families where the in vivo tissue, developmental, and substrate specificity of most members has still to be established.

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The flavour precursors and their role

Four non-volatile, odourless CSOs (Table 1) are the precursors of the flavour and odours of the Alliums, but just the first three of them are characteristic for Allium sativum. 2

Table 1. Precursors of the flavour and odours of the Genus Allium

Besides them, several γ-glutamyl peptide (γGP) derivatives of these flavour compounds have been detected. Over 17 types have been isolated and including γ-glutamyl-S-alk(en)yl glutathiones, γ-glutamyl-S-alk(en)yl cysteines and γ-glutamyl-S-alk(en)yl cysteine sulphoxides, all proposed to derive from glutathione (γ-glutamyl cysteinyl glycine). Although they do not appear to contribute directly to flavour, the current view is that they are intermediates in biosynthesis and may also act as reserves of nitrogen and sulphur.

The levels of non-protein cysteine and glutathione derivatives amount to 15% dry weight, indicating that byosynhtesis of flavour precursors is a major biosynthetic activity within the plant. The two roles that have been ascribed are for defence against pests and predation, particularly in the overwintering bulb, and for carbon, nitrogen, and sulphur storage and transport. 3

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Byosynhtesis of flavour precursors

Lancaster and colleagues 4 have proposed a pathway requiring γGPs as intermediates, which has become accepted as the biosynthetic route. It proposes (Figure 1A) that the biosynthesis of the flavour precursors in garlic proceeds via S-alk(en)ylation of the cysteine in glutathione, followed by transpeptidation to remove the glycyl group, oxidation to the cysteine sulphoxide, and, finally, removal of the glutamyl group to yield CSOs. An alternative biosynthetic route (Figure 1B) omits glutathione in favour of direct alk(en)ylation of cysteine or thioalk(en)ylation of O-acetyl serine followed by oxidation to a sulphoxide.

Figure 1. Biosynthetic pathways(A) and (B) proposed by Lancaster et al. for the formation of flavour precursors

The relative contribution of both pathways in all tissues and throughout the life history is also unclear and the source of the alk(en)yl groups remains to be resolved. In this work we describe the pathways of Figure 1. analysing these steps:

Sulphur assimilation

Synthesis of cysteine

Origin of the alk(en)yl substituents

Participation of glutathione

Oxidation step

Role of γ-glutamil-S-transpeptidase



Sulphur assimilation

Sulphur 5 is taken up from the soil by the roots as sulphate (SO42- ), most of which is transported in the xylem to the leaf tissue where it is reduced to sulphide and assimilated into cysteine in light-dependant reactions. It should be noted however that some reduction of sulphate and assimilation into cysteine can take place in the roots.

Figure 2. Summary of reduction and assimilation of sulphur in plants

Metabolism is initiated by an adenylation reaction catalyzed by ATP sulfurylase and the reaction product 5'-adenylylsulfate (APS) is a branch point intermediate, which can be channeled toward reduction or sulfation:

Reduction is carried out in two steps. In the first step, APS reductase transfers two electrons to APS to produce sulfite. The best evidence so far is that the electrons are derived from glutathione (GSH):

In the second reaction, sulfite reductase (1.8.7.1) transfers 6 electrons from ferredoxin to produce sulphide:

These steps can be summed up as follow:



Synthesis of cysteine

The final step of sulphur assimilation consists of cysteine synthesis from sulphide and serine. This reaction is catalysed by two consecutive enzymes, serine acetyltransferase or SAT and cysteine synthase or CS (or OAS thiol lyase). It is the stage at which inorganic sulphur is incorporated into the first organic sulphur compound within the cell and is also the point at which the carbon and nitrogen assimilation pathways meet sulphur assimilation. 2

Serine acetyltransferase (2.3.1.30) (a)
Cysteine synthase (2.5.1.47) (b)

After addition of an O-acetyl group to serine by SAT, a new adduct of O-acetylserine is formed, bound to the pyridoxal-5-phosphate co-factor of CS:

This reacts with the second substrate, sulphide, to form cysteine which is released:

This process can be summed up as follow:

O-acetylserine or cysteine can react directly with an alk(en)yl donor as shown in pathway (B) of Figure 1.



Origin of the alk(en)yl substituents

Methacrylate, proposed as the precursor of the allyl, propyl, and propenyl groups occurs within the cell during the breakdown of the branched chain fatty acid valine, probably within the plant peroxisome. It was found 1 that methacrylate is an intermediate in valine catabolism thanks to experiments with radiolabelled 14C-valine. The suggestion from these data was that all the alk(en)yl side-chains in the CSOs (except methyl) were derived from a S-2-carboxypropyl group through decarboxylation and reduction as indicated in Figure 3.

Figure 3. Proposed pathway for the byosinthesys of the allyl, propyl, and propenyl groups

A further example of a possible origin for the allyl group characteristic of garlic comes from the over 120 glucosinolate secondary metabolites found in some variety of Allium, but this topic is not discussed in this work.



Participation of glutathione

Glutathione (GSH) is synthesized in both cytosol and chloroplast and this is co-ordinated with the availability of cysteine. GSH homeostasis is a complex interplay of synthesis, transport, storage, oxidation/reduction, further metabolism, and catabolism and many aspects of these processes are poorly understood.

2D and 3D (c) structures of Glutathione.

The enzyme glutathione-S-transferase (GST) it has been detected in the epidermal tissue from Alliums bulbs and its activity has not been studied in detail yet. As it was present in the cytosolic as well as microsomal fractions, it means that the enzymes in each compartment has a different specificity. The GSTs are a large and ancient enzyme superfamily, where the N-terminal glutathione binding site is better conserved than the C-terminal cosubstrate binding site.

GST catalyze the reaction of the thiol group on GSH with a wide range of hydrophobic and electrophilic substances. The GSTs are integral to the mechanisms by which plants and animals defend themselves against xenobiotics, such as herbicides, through conjugation of the toxin with glutathione to increase its water solubility.

Glutathione-S-transferase(2.5.1.18) (d)

The role of GSH in the metabolic pathway of flavour precursors can be summed up as follow:

  1. GST links the thiol group of GSH to an alk(en)yl donor (in Figure 1A a S-methyl donor)
  2. another enzyme (maybe a carboxypeptidase) remove the glycine
  3. a γ-glutamyl alk(en)yl cysteine is left


Oxidation step

Evidence for the general nature of the oxidation step comes from several studies that demonstrate oxidation of exogenous S-alk(en)yl cysteines to the corresponding sulphoxide. So S-methyl cysteine, S-propyl cysteine, and S-propenyl cysteine could all be oxidized to the corresponding sulphoxide by leaf tissue. More recent experiments reinforce this finding. The oxidase proposed by Lancaster and Shaw 6 to act on γ-glutamyl-S-alk(en)yl cysteines may also recognize S-alk(en)yl cysteines.



Role of γ-glutamil-S-transpeptidase

The relationship between the γ-glutamyl peptides and alk(en)yl cysteine sulphoxides requires the activity of enzymes to remove the glycyl and γ-glutamyl residues from the nascent alk(en)yl sulphoxide. 7 γ-Glutamyl transpeptidase catalyses the transfer of the γ-glutamyl group from γglutamyl peptides to either amino acids or other peptides. This enzyme may also act as a γ-glutamyl peptidase, requiring only water as an acceptor. The enzyme activities were higher in the rapidly growing leaves.

A γ-glutamyl transpeptidase was partially purified from onion (Lancaster and Shaw, 1994) that exhibited both peptidase activity, which was independent of pH, and transpeptidase activity that increased substantially above pH 8.0. It exhibited Km values between 0.4 mM and 2.0 mM for several γ-glutamyl derivatives and a Km value for glutathione of 5 mM. It has a broad substrate specificity including γ-glutamyl methyl cysteine, γ-glutamyl propenyl cysteine, 2-carboxyglutathione, and γ-glutamyl propenyl cysteine sulphoxide.

γ-glutamyl transpeptidase (2.3.2.2) (e)

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Byosynhtesis of organoselenium compounds

One final example of synthesis of compounds analogous to the Allium flavour precursors originates from the mechanism of selenium tolerance found in some plants. Sulphur and selenium are chemically similar, and selenium can thus be incorporated into compounds that should contain sulphur, leading to toxicity problems. Analysis of selenium-containing volatiles from onion, garlic, A. tuberosum, and A. ampeloprasum L. has shown that Se-methyl compounds predominate despite the abundance of other S-alk(en)yl compounds in the plants. Discrimination between the two elements occurs in some situations and one of them is in the synthesis of Se-methyl selenocysteine in the tolerance mechanism of plants within the Fabaceae that accumulate high levels of selenium. These plants synthesize Se-methyl selenocysteine, γ-glutamyl-Se-methyl selenocysteine, and other non-protein amino acid selenium derivatives, which could be considered the selenium analogues of S-alk(en)yl cysteines and γ-glutamyl-alk(en)yl cysteine peptides.

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