The Biosynthesis of Alliin

1. The Biosynthesis of Alliin Jill Maria Hughes with Hamish Collin, Rick Cosstick, Meriel Jones, Brian Tomsett and Angela Tregova EU Project No.QLK1-CT-1999-00498

2. Biochemistry of Garlic at Liverpool • How is Alliin synthesised • Where is it synthesised? • When is it synthesised?

3. SO42- SO32- SO22- cysteine Biosynthetic Pathway valine & methacrylate serine Allyl group (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 S-trans-1-propenylcysteine sulphoxide (isoalliin) methiin alliin S-allyl-cysteine sulphoxide (alliin)

4. Cysteine Unknown Allyl source Serine Glutathione Allyl Glutathione Serine pathway Gamma-Glutamyl Allyl Cysteine AllylCysteine Glutathione pathway Alliin The Serine and Glutathione Pathways

5. Our Approach this year Precursor feeding. Modify method from isocratic to gradient. Use HPLC to identify intermediates. Allyl Cysteine Synthase. Identification of a specific garlic enzyme that can synthesise Allyl Cysteine.

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

7. HPLC Method Development Gamma-glutamylallylcysteine Alliin Isoalliin Typical HPLC trace of Garlic clove gradient

8. HPLC Method Development Peak Identification: • Compare unknown peak retention times with standards • Mass spectroscopy-identify from ‘general profile’ and mass of molecular ion

9. HPLC Method Development Gamma-glutamylallylcysteine Alliin Gamma-glutamylisoallylcysteine Isoalliin Typical HPLC trace of Garlic clove gradient

10. Precursor Feeding Experiments Garlic Using single clone garlic callus to reduce variation In Allium callus tissue the general reaction seems to apply: Alk(en)yl thiol Alk(en)yl cysteine Alk(en)yl CSO Onion

11. Precursor Feeding Experiments- 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) Alliin 100 Allyl Cysteine Allyl thiol 80 60 40 Onion callus incubated with allyl thiol and propyl thiol for six days. 20 0 0.00 10.00 20.00 30.00 40.00 Propiin Propyl cysteine 100 Propyl thiol 80 60 40 20 0 0.00 10.00 20.00 30.00 40.00 100 80 Control 60 40 20 0 0.00 10.00 20.00 30.00 40.00

12. Cysteine synthase Sulphide + O-Acetyl Serine Cysteine Allyl thiol + O-Acetyl Serine Allyl Cysteine Alliin Allyl Cysteine synthase 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?

13. The Serine and Glutathione Pathways Cysteine Serine Unknown Allyl source Glutathione Allyl Glutathione Serine pathway Allyl Cysteine synthase Gamma-Glutamyl Allyl Cysteine AllylCysteine Glutathione pathway Alliin

14. Protein purification:Ion Exchange chromatography Garlic leaves were fractionated with ammonium sulphate then separated by ion-exchange chromatography. Many fractions show cysteine synthase activity Only a few fractions show allyl cysteine synthase activity

15. Protein purification: Hydrophobic Interaction Chromatography Allyl cysteine synthase and cysteine synthase activity co-elute Cysteine production was assayed colorimetrically and allyl cysteine by HPLC

16. 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 34000 Fractions 26 27 28 29 30

17. 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)

18. 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?

19. The Serine and Glutathione Pathways Cysteine Serine Unknown Allyl source Glutathione Glutathione-S-Transferase Allyl Glutathione Serine pathway Allyl Cysteine synthase Gamma- Glutamyl Allyl Cysteine AllylCysteine Glutathione pathway Alliin

20. The Biosynthesis of Alliin Jill Maria Hughes Acknowledgements: Mark Prescott Mass Spectroscopy Mark Wilkinson Protein purification facilities and Peptide Sequencing EU Project No.QLK1-CT-1999-00498