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

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

Biosynthetic Pathway SO 4 2- SO 3 2- SO 2 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-propenylcysteine oxidase 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 & methacrylate serine oxidase S-allylcysteine S-allyl-cysteine sulphoxide (alliin)

The Serine and Glutathione Pathways Allyl Cysteine Alliin Serine pathway Serine Unknown Allyl source CysteineAllyl Glutathione Gamma-Glutamyl Allyl Cysteine Glutathione pathway Glutathione

Our Approach this year Precursor feeding. Modify method from isocratic to gradient. Use HPLC to identify intermediates. Allyl Cysteine Synthase. Allyl Cysteine Synthase. Identification of a specific garlic enzyme that can 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 Alliin Isoalliin Gamma - glutamylallylcysteine Typical HPLC trace of Garlic clove gradient

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

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

Precursor Feeding Experiments 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 Onion callus incubated with allyl thiol and propyl thiol for six days. Allyl thiol Propyl thiol Alliin Allyl Cysteine PropiinPropyl 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 SerineUnknown Allyl source Cysteine Allyl Glutathione Glutathione pathway Glutathione Allyl Cysteine synthase Gamma-Glutamyl Allyl Cysteine

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

What is the Enzyme? The selected 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 SerineUnknown Allyl source Cysteine Allyl Glutathione Gamma- Glutamyl Allyl Cysteine Glutathione pathway Glutathione Allyl Cysteine synthase Glutathione-S- Transferase

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