The Biosynthesis of Alliin

Jill Maria HughesJill Maria Hughes

EU Project No.QLK1-CT-1999-00498

with Hamish Collin, Rick Cosstick, Meriel Jones, Brian Tomsett and Angela

Tregova

Biochemistry of Garlic at Liverpool

How is Alliin synthesisedHow is Alliin synthesised

Where is it synthesised?When is it synthesised?

Biosynthetic PathwaySO4

2- SO32- SO2

2- cysteine

glutathione(γ-glu-cys-gly)

S-methyl-γ-glu-cys

gly

S-methylcysteine

methiin

glu

trans-peptidase

oxidase

S-2-CP-γ-glu-cys

gly

S-trans-1-propenyl-γ-glu-cys

S-trans-1-propenylcysteineoxidase

trans-peptidase

glu

HCOOH

S-trans-1-propenylcysteine sulphoxide(isoalliin)

S-methylglutathioneS-(2-carboxypropyl)-glutathioneS-allylglutathione

S-allyl-γ-glu-cys

gly

S-allylcysteine

glu

trans-peptidase

oxidase

alliin

Allyl group(unknown sources)

valine & methacrylateserine

oxidase

S-allylcysteine

S-allyl-cysteine sulphoxide(alliin)

The Serine and Glutathione Pathways

Allyl Cysteine

 Alliin

Serine pathway

SerineUnknown Allyl source

Cysteine

Allyl Glutathione

Gamma-Glutamyl Allyl Cysteine

Glutathione pathway

Glutathione

Our Approach this year

Precursor feeding. Precursor feeding. Modify method from isocratic to gradient.Modify method from isocratic to gradient.Use HPLC to identify intermediates.Use HPLC to identify intermediates.

Allyl Cysteine Synthase.Allyl Cysteine Synthase.Identification of a specific garlic enzyme that can Identification of a specific garlic enzyme that can

synthesise Allyl Cysteine.synthesise Allyl Cysteine.

HPLC Method Development

•Heptane sulphonic acid method was tried but was not suitable for indentification of compounds by Mass Spectroscopy

•We developed a simple HCl:Acetonitrile gradient which allows simultaneous identification of cysteine sulphoxides and gamma-glutamyl peptides.

•This method is now in routine use in our laboratory.

To aid identification of cysteine conjugates, our simple isocratic HPLC Method has been adapted:

HPLC Method Development

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Alliin

Isoalliin

Gamma-glutamylallylcysteine

Typical HPLC trace of Garlic clove

gradient

HPLC Method Development

•Compare Compare unknown peak unknown peak retention times retention times with standardswith standards

•Mass spectroscopy-identify from ‘general profile’ and mass of molecular ion

Standards Rt 0.9ml/min

Methyl CSO 4.25Ethyl CSO 3.76Methyl cysteine 4.25Alliin 4.49Propenyl CSO 5.24Propyl CSO 5.76Glutathione 6.01Gamma glutamyl cysteine 6.78Methionine 6.74Ethyl cysteine 7.04Methyl glutathione 10.113-CPC 10.562-CPC 11.74Allyl cysteine 11.69Butyl CSO 12.061,2 Dicarboxy glutathione 12.53Propenyl cysteine 14.66propyl cysteine 15.44Gamma glutamyl allyl cysteine 20.19Butyl cysteine 21.86Propyl glutathione 20.8

Synergi RPMax 100%0.03MHCl@5min 50%Acetonitrile@25min

Peak Identification:Peak Identification:

HPLC Method Development

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Alliin

Isoalliin

Gamma-glutamylallylcysteine

Gamma-glutamylisoallylcysteine

Typical HPLC trace of Garlic clove

gradient

Precursor Feeding ExperimentsGAt2b GPt2a GAC2b GPC2c Av GBl2

Alliin 41 42 509 6 14Propiin 12 135 14 26 3Allyl cysteine 81 0 198 0 0Propyl cysteine 5 85 12 138 0

GAt6b GPt6c GAC6a GPC6a Av GBl6Alliin 0 56 22 24 20Propiin 0 239 14 61 4Allyl cysteine 36 34 348 27 0Propyl cysteine 0 161 8 248 6

GAt13a GPt13b GAC13b GPC13b Av GBl13Alliin 0 23 53 30 14Propiin 0 203 6 167 12Allyl cysteine 0 0 302 0 0Propyl cysteine 0 60 0 254 11

OnAt2c OnPt2a OnAC2a OnPC2b Av OnBl2Alliin 146 12 172 80 77Propiin 9 31 27 30 10Allyl cysteine 196 0 446 0 0Propyl cysteine 23 80 25 170 14

OnAt6c OnPt6a OnAC6a OnPC6b Av OnBl6Alliin 131 74 586 109 88Propiin 24 103 9 325 13Allyl cysteine 286 65 716 0 0Propyl cysteine 23 163 0 278 30

OnAt13c OnPt13b OnAC13b OnPC13b Av OnBl13Alliin 34 12 348 57 53Propiin 12 106 11 171 16Allyl cysteine 153 0 349 0 0Propyl cysteine 9 21 8 188 15

Onion

Garlic

In Allium callus tissue the general reaction seems to apply:

Alk(en)yl thiol

Alk(en)yl cysteine

Alk(en)yl CSO

Using single clone garlic callus to reduce variation

Precursor Feeding Experiments-

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Onion callus incubated with allyl thiol and propyl thiol for six days.

Allyl thiol

Propyl thiol

AlliinAllyl Cysteine

Propiin Propyl cysteine

Both Garlic and Onion callus can convert exogenously applied allyl thiol and propyl thiol to their respective cysteine conjugate and subsequently to their respective cysteine sulphoxide(CSO)

Control

Is there an Allyl Cysteine Synthase?

Some purified plant cysteine synthase enzymes have shown the capability of producing cysteine conjugates in addition to their normal function

Is there a specific cysteine synthase homologue in garlic that can conjugate allyl thiol to (O-acetyl)serine to give allyl cysteine and subsequently alliin?

Sulphide + O-Acetyl Serine Cysteine

Allyl thiol + O-Acetyl Serine Allyl Cysteine Alliin

Cysteine synthase

Allyl Cysteine synthase

The Serine and Glutathione Pathways

Allyl Cysteine

 Alliin

Serine pathway

Serine Unknown Allyl source

Cysteine

Allyl Glutathione

Glutathione pathway

Glutathione

Allyl Cysteine synthase Gamma-

Glutamyl Allyl Cysteine

Cysteine synthase activity. Q-Sepharose. 7.11.01

00.05

0.10.15

0.20.25

0.3

Fraction

OD

cysteine synthaseactivity

Many fractions show cysteine synthase activity

Protein purification:Ion Exchange chromatography

Garlic leaves were fractionated with ammonium sulphate then separated by ion-exchange chromatography.

Only a few fractions show allyl cysteine synthase activity

Protein purification: Hydrophobic Interaction Chromatography

Allyl cysteine synthase and cysteine synthase activity co-elute

Phenyl sepharose fractionation

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23

25

27

29

31

33

35

37

39

Fraction

OD

55

0

cysteinesyntase activity

allyl cysteinesynthaseactivity

Cysteine production was assayed colorimetrically and allyl cysteine by HPLC

Protein purification: How pure is the Fraction?

 

SDS-PAGE shows a distinct band in the allyl cysteine synthase active fractions at approx. 34000 kdal.

This M Wt. is consistent with plant cysteine synthase monomers found previously

Fractions 26 27 28 29 30

34000

What is the Enzyme?

The selected 34000 band was digested with trypsin and the resultant peptides separated by preparative HPLC.

Three selected peptides were sequenced:-

…….FLGVMPSHYSIE………. YLGADLALTDTN………… SANPGAHYATTGP………….

A simple BLAST search of these peptides in the protein database shows most similarity to a cysteine synthase from Oryza sativa (Rice)

Biochemistry of Garlic at Liverpool

Where now?

This enzyme will be selected and overexpressed

Enyme kinetics of the purified enzyme should help to clarify it’s role in vivo.

The future?

Use similar techniques to probe the glutathione pathway?

The Serine and Glutathione Pathways

Allyl Cysteine

 Alliin

Serine pathway

Serine Unknown Allyl source

Cysteine

Allyl Glutathione

Gamma- Glutamyl Allyl Cysteine

Glutathione pathway

Glutathione

Allyl Cysteine synthase

Glutathione-S-Transferase

The Biosynthesis of Alliin

Jill Maria HughesJill Maria Hughes

Acknowledgements:

Mark Prescott Mass Spectroscopy

Mark Wilkinson Protein purification facilities and Peptide SequencingEU Project No.QLK1-CT-1999-00498