Transcript WP4 - Hughes

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
Biochemistry of Garlic at Liverpool

How is Alliin synthesised
Where
is it synthesised?
When is it synthesised?
Biosynthetic Pathway
SO42serine
SO32-
Allyl group
(unknown sources)
SO22valine &
methacrylate
S-allylglutathione
cysteine
glutathione
(γ-glu-cys-gly)
S-(2-carboxypropyl)-glutathione
gly
S-allyl-γ-glu-cys
S-methylglutathione
gly
S-2-CP-γ-glu-cys
gly
S-methyl-γ-glu-cys
HCOOH
glu
transpeptidase
S-allylcysteine
S-allylcysteine
oxidase
oxidase
alliin
S-allyl-cysteine sulphoxide
(alliin)
S-trans-1-propenyl-γ-glu-cys
glu
transpeptidase
glu
transpeptidase
S-methylcysteine
S-trans-1-propenylcysteine
oxidase
S-trans-1-propenylcysteine sulphoxide
(isoalliin)
oxidase
methiin
The Serine and Glutathione Pathways
Cysteine
Unknown
Allyl source
Serine
Glutathione
Allyl
Glutathione
Serine
pathway
Gamma-Glutamyl
Allyl Cysteine
Allyl Cysteine
Alliin
Glutathione
pathway
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.
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 gammaglutamyl peptides.
•This method is now in routine
use in our laboratory.
HPLC Method Development
Alliin
Gamma-glutamylallylcysteine
Isoalliin
Typical HPLC
trace of Garlic
clove
gradient
HPLC Method Development
Standards
Peak Identification:
•Compare unknown
peak retention times
with standards
•Mass spectroscopyidentify from ‘general
profile’ and mass of
molecular ion
Methyl CSO
Ethyl CSO
Methyl cysteine
Alliin
Propenyl CSO
Propyl CSO
Glutathione
Gamma glutamyl cysteine
Methionine
Ethyl cysteine
Methyl glutathione
3-CPC
2-CPC
Allyl cysteine
Butyl CSO
1,2 Dicarboxy glutathione
Propenyl cysteine
propyl cysteine
Gamma glutamyl allyl cysteine
Butyl cysteine
Propyl glutathione
Rt 0.9ml/min
4.25
3.76
4.25
4.49
5.24
5.76
6.01
6.78
6.74
7.04
10.11
10.56
11.74
11.69
12.06
12.53
14.66
15.44
20.19
21.86
20.8
Synergi RPMax 100%0.03MHCl@5min
50%Acetonitrile@25min
HPLC Method Development
Alliin
Gamma-glutamylallylcysteine
Gamma-glutamylisoallylcysteine
Isoalliin
Typical HPLC
trace of Garlic
clove
gradient
Precursor Feeding Experiments
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
Garlic
GAt2b
Alliin
Propiin
Allyl cysteine
Propyl cysteine
GPt2a
41
12
81
5
GAt6b
Alliin
Propiin
Allyl cysteine
Propyl cysteine
GPt6c
0
0
36
0
GAt13a
Alliin
Propiin
Allyl cysteine
Propyl cysteine
42
135
0
85
GAC6a
56
239
34
161
GPt13b
0
0
0
0
GAC2b
GPC2c
Av GBl2
509
6
14
14
26
3
198
0
0
12
138
0
23
203
0
60
22
14
348
8
GPC6a
24
61
27
248
Av GBl6
20
4
0
6
GAC13b GPC13b Av GBl13
53
30
14
6
167
12
302
0
0
0
254
11
Onion
Alliin
Propiin
Allyl cysteine
Propyl cysteine
OnAt2c
OnPt2a
OnAC2a OnPC2b Av OnBl2
146
12
172
80
77
9
31
27
30
10
196
0
446
0
0
23
80
25
170
14
Alliin
Propiin
Allyl cysteine
Propyl cysteine
OnAt6c
OnPt6a
OnAC6a OnPC6b Av OnBl6
131
74
586
109
88
24
103
9
325
13
286
65
716
0
0
23
163
0
278
30
Alliin
Propiin
Allyl cysteine
Propyl cysteine
OnAt13c OnPt13b OnAC13b OnPC13b Av OnBl13
34
12
348
57
53
12
106
11
171
16
153
0
349
0
0
9
21
8
188
15
Precursor Feeding ExperimentsBoth 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
80
Allyl Cysteine
Allyl thiol
60
40
Onion callus
incubated with
allyl thiol and
propyl thiol for
six days.
20
0
0.00
100
10.00
20.00
Propiin
80
30.00
Propyl
cysteine
60
40.00
Propyl
thiol
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
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?
Cysteine synthase
Sulphide + O-Acetyl Serine
Cysteine
Allyl thiol + O-Acetyl Serine
Allyl Cysteine
Allyl Cysteine synthase
Alliin
The Serine and Glutathione Pathways
Cysteine
Serine
Serine
pathway
Unknown Allyl source
Glutathione
Allyl
Glutathione
Allyl
Cysteine
synthase
Allyl Cysteine
Alliin
Gamma-Glutamyl
Allyl Cysteine
Glutathione
pathway
Protein purification:
Ion Exchange chromatography
Garlic leaves were fractionated with ammonium sulphate
then separated by ion-exchange chromatography.
Cysteine synthase activity.
Q-Sepharose. 7.11.01
0.3
0.25
0.2
0.15
0.1
0.05
0
Fr
1
Fr
5
Fr
9
Fr
13
Fr
17
Fr
21
Fr
25
Fr
29
Fr
33
Only a few fractions show
allyl cysteine synthase
activity
OD
Many fractions show
cysteine synthase
activity
cysteine synthase
activity
Fraction
Protein purification:
Hydrophobic Interaction Chromatography
0.6
0.5
0.4
0.3
0.2
cysteine 0.1
syntase activity
Fraction
39
37
35
33
31
29
27
25
23
21
19
17
15
13
11
9
7
5
allyl cysteine
synthase
activity
3
0
1
Cysteine production
was assayed
colorimetrically and
allyl cysteine by HPLC
0.7
OD550
Allyl cysteine
synthase and
cysteine synthase
activity co-elute
Phenyl sepharose fractionation
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
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)
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
Cysteine
Serine
Serine
pathway
Unknown Allyl source
Glutathione
Allyl
Glutathione
Allyl
Cysteine
synthase
Allyl Cysteine
Alliin
Glutathione-STransferase
Gamma- Glutamyl
Allyl Cysteine
Glutathione
pathway
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