Transcript WP4 - Hamish Collin

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