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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 SO42serine SO32- SO22- cysteine valine & methacrylate allyl (unknown sources) S-allylglutathione glutathione (γ-glu-cys-gly) S-(2-carboxypropyl)-glutathione S-methylglutathione gly S-allyl-γ-glu-cys gly S-2-CP-γ-glu-cys gly S-methyl-γ-glu-cys HCOOH glu glu transpeptidase S-allylcysteine S-trans-1-propenyl-γ-glu-cys glu transpeptidase S-allylcysteine transpeptidase S-methylcysteine S-trans-1-propenylcysteine oxidase oxidase alliin S-allyl-cysteine sulphoxide (alliin) oxidase oxidase S-trans-1-propenylcysteine sulphoxide methiin (isoalliin) 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 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 Substrate feeding to callus Results: alliin allyl cysteine isoalliin 10,1;10,1 10; 10,1 allyl thiol propyl thiol allyl cysteine 10;10,1 propenyl cysteine propyl cysteine propiin propyl cysteine 10; 1;10 1;10,1 10,1;10,1 Incubation for 5 days with 10mM or 1mM substrate Incubation for 12/15 days with 10mM or 1mM substrate 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 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 in vivo 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 Cysteine synthase 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 + Allyl thiol Ac-O-CH2CH(COOH)(NH2) O-acetyl-serine ( or H-S-CH2CH(COOH)(NH2) ) cysteine CH2=CH-CH2-S-CH2CH(COOH)(NH2) Allyl cysteine 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? 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. cysteine synthesis by Q-Sepharose column fractions 80.0 60.0 40.0 20.0 9. 0 11 .0 13 .0 15 .0 17 .0 19 .0 7. 0 5. 0 3. 0 0.0 1. 0 uM cysteine 100.0 Fraction