Garlic

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

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

CSO 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 (unknown sources) valine & methacrylate serine oxidase S-allylcysteine S-allyl-cysteine sulphoxide (alliin)

Substrate feeding to garlic callus zGarlic 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 z 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: ythe substrate gets into the cell yenzymes are present that utilise the substrate ythe product is not further metabolised

Compounds fed to callus z*Allyl thiol z*Propyl thiol z*Allyl alcohol z*Propyl alcohol zAllyl cysteine zPropyl cysteine zPropenyl cysteine z*Methacrylic acid z*Vinyl acetic acid z2-Carboxypropyl cysteine z3-Carboxypropyl cysteine zCysteine zSerine zGlutathione zCystathionine * with and without cysteine and serine

Substrate feeding to callus 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 Results: 2CPC has also been shown to produce isoalliin

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 zIs there a enzyme in garlic, an allyl cysteine synthase enzyme, which can synthesise allyl cysteine from allyl thiol zIf present, does this synthesis occur in vivo zThis enzyme is being pursued in two ways: yby protein purification with appropriate assays yby looking directly at the genes, using known cysteine synthase genes

Cysteine synthase Cysteine synthase is an enzyme that makes cysteine H-S-H + Ac-O-CH 2 CH(COOH)(NH 2 ) Reduced Sulphur O-acetyl-serine H-S-CH 2 CH(COOH)(NH 2 ) cysteine Some purified cysteine synthase enzymes can also make allyl cysteine from allyl thiol CH 2 =CH-CH 2 -S-H + Ac-O-CH 2 CH(COOH)(NH 2 ) Allyl thiol O-acetyl-serine ( or H-S-CH 2 CH(COOH)(NH 2 ) ) cysteine CH 2 =CH-CH 2 -S-CH 2 CH(COOH)(NH 2 ) Allyl cysteine

Possible mechanisms ? zIs allyl thiol present? zWhere does the allyl moiety come from? zDoes the sulphur atom derive from attachment to the allyl group or to a ß-substituted alanine group?

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.