Garlic

1. Garlic

2. The Biochemical Synthesis of ‘Alliin’ in Garlic Hamish Collin School of Biological Sciences University of Liverpool Jill Hughes, Brian Tomsett, Meriel Jones, Rick Cosstick & Angela Tregova

3. Garlic Research at Liverpool Up to now: Chemical synthesis of standards Substrate feeding to garlic callus Differential Display In Progress: Cysteine synthase enzyme To be done: Labeled substrate feeding

4. SO42- SO32- SO22- cysteine CSO biosynthetic pathway valine & methacrylate serine allyl (unknown sources) glutathione (γ-glu-cys-gly) S-allylglutathione S-(2-carboxypropyl)-glutathione S-methylglutathione gly gly gly S-allyl-γ-glu-cys S-2-CP-γ-glu-cys S-methyl-γ-glu-cys HCOOH glu glu S-trans-1-propenyl-γ-glu-cys trans- peptidase trans- peptidase trans- peptidase glu S-allylcysteine S-methylcysteine S-allylcysteine S-trans-1-propenylcysteine oxidase oxidase oxidase oxidase alliin S-trans-1-propenylcysteine sulphoxide (isoalliin) methiin S-allyl-cysteine sulphoxide (alliin)

5. Substrate feeding to garlic callus • Garlic callus has been previously shown to be capable of limited synthesis of flavour precursors indicating that at least some of the enzyme systems were present. The absence of a cuticle ensured a greater chance of uptake of pathway intermediates • A range of potential intermediates on the pathway of CSO production have been synthesised and purchased, have been fed to garlic callus and the products identified (where possible) by HPLC. This method of substrate feeding will only give a positive result if: • the substrate gets into the cell • enzymes are present that utilise the substrate • the product is not further metabolised

6. Compounds fed to callus • *Allyl thiol • *Propyl thiol • *Allyl alcohol • *Propyl alcohol • Allyl cysteine • Propyl cysteine • Propenyl cysteine • *Methacrylic acid • *Vinyl acetic acid • 2-Carboxypropyl cysteine • 3-Carboxypropyl cysteine • Cysteine • Serine • Glutathione • Cystathionine * with and without cysteine and serine

7. Substrate feeding to callus Results: 2CPC has also been shown to produce isoalliin Conclusion: These experiments suggest that in vivo the general reaction shown may occur:- Alk(en)yl thiol Alk(en)yl cysteine Alk(en)yl CSO

8. Cysteine synthase There is evidence in the literature that some purified cysteine synthase type enzymes are multifunctional and amongst other reactions can take allyl thiol and attach it to an amino acid skeleton to make Allyl cysteine Some other ß-substituted alanines (secondary plant products such as mimosine) are known to be synthesised by cysteine synthase isoenzymes Question • Is there a enzyme in garlic, an allyl cysteine synthase enzyme, which can synthesise allyl cysteine from allyl thiol • If present, does this synthesis occur invivo • This enzyme is being pursued in two ways: • by protein purification with appropriate assays • by looking directly at the genes, using known cysteine synthase genes

9. Cysteine synthase is an enzyme that makes cysteine H-S-H + Ac-O-CH2CH(COOH)(NH2) Reduced Sulphur O-acetyl-serine H-S-CH2CH(COOH)(NH2) cysteine Some purified cysteine synthase enzymes can also make allyl cysteine from allyl thiol CH2=CH-CH2-S-H + Ac-O-CH2CH(COOH)(NH2) Allyl thiol O-acetyl-serine ( or H-S-CH2CH(COOH)(NH2) ) cysteine CH2=CH-CH2-S-CH2CH(COOH)(NH2) Allyl cysteine Cysteine synthase

10. Possible mechanisms ? • Is allyl thiol present? • Where does the allyl moiety come from? • Does the sulphur atom derive from attachment to the allyl group or to a ß-substituted alanine group?

11. Cysteine synthase purification • Aim: • To partially purify a garlic allyl cysteine synthesising enzyme • obtain amino acid sequence data • compare data to the garlic cysteine synthase enzyme sequences being obtained by molecular biology techniques • Garlic leaf tissue has been fractionated by ammonium sulphate precipitation and ‘cysteine synthase active’ fractions applied to a Q-Sepharose column. Active fractions are currently being assayed.