S. Patterson, D. O’Hagan / Phytochemistry 61(2002) 323–329
329
Bakola-Christianopoulou et al., 1994). Mass enrich-
ments of isolated littorine 1 and hyoscyamine 2 were
calculated by comparison with unlabelled samples
[commercial hyoscyamine, Aldrich Chemical Co., and
littorine 1 prepared as previously described (Robins et
al., 1994)].
Chesters, N.C.J.E., O’Hagan, D., Robins, R.J., Kastelle, A., Floss,
H.G., 1995. The biosynthesis of tropic acid—the stereochemical
course of the mutase involved in hyoscyamine biosynthesis in
Datura stramonium. Chem. Commun. 129–130.
Chesters, N.C.J.E., Walker, K., O’Hagan, D., Floss, H.G., 1996. The
biosynthesis of tropic acid: A reevaluation of the stereochemical
course of the conversion of phenyllactate to tropate in Datura stra-
monium. J. Am. Chem. Soc. 118, 925–926.
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mechanism of the cobamide coenzyme dependent isomerisation of
methylmalonyl CoA to succinyl CoA. J. Am. Chem. Soc. 82, 2643–
2644.
4.7. Preparation of cell free extract and a
dehydrogenase assay
All procedures were carried out at 4 ꢀC. The cell
masses from six root cultures of D. stramonium aged 11–
17 days (fresh wt. 29.4 g) were harvested, washed with
cold water and blotted dry. The cells were then frozen in
liquid N2 and ground in a pre-chilled pestle and mortar
with sand and extraction buffer (60 ml, see later) for 20
min. The slurry was filtered through 3 layers of cheese-
cloth and clarified by centrifugation (10,000 Â g, 30
min). The supernatant protein solution was then used as
the crude extract for enzyme assays.
Humphrey, A.J., O’Hagan, D., 2001. Tropane alkaloid biosynth-
esis. A century old problem unresolved. Nat. Prod. Rep. 18,
494–502.
Kilgore, J.L., Aberhart, D.J., 1991. Lysine 2,3-aminomutase: role of
S-adenosyl-l-methionine in the mechanism. Demonstartion of tri-
tium transfer from (2RS,3RS)-[3-3H]-Lysine to S-adenosyl-l-
methionine. J. Chem. Soc., Perkin Trans. 1, 79–84.
Leete, E., 1987. Stereochemistry of the 1,2-migration of the carboxyl
group that occurs during the biosynthesis of tropic acid from phe-
nylalanine. Can. J. Chem. 65, 226–228.
Leete, E., Kowanko, N., Newmark, R.A., 1975. Use of carbon-13
nuclear magnetic resonance to establish that the biosynthesis of
tropic acid involves an intramolecular rearrangement of phenylala-
nine. J. Am. Chem. Soc. 97, 6826–6830.
Extraction buffer: Phosphate buffer (100 mM) pH 7.5
containing; glycerol (20%), DTT (5 mM), cysteine
(0.05%), b-mercaptoethanol (10 mM), FAD (5 mM),
FMN (5 mM) and sodium ascorbate (50 mM).
Dehydrogenase assay. In a cuvette (1.5 ml) was added
0.2 M Tris–HCl buffer (1.4 ml, pH 7.3), NAD or NADP
(50 ml of 6.6 mM) and hyoscyamine 2 (50 ml of 30 mM).
This mixture was incubated for 5 min before the addi-
tion of 100 ml of cell free extract and the absorbance
recorded at 340 nm over 10 min. No change in absor-
bance was observed.
McKenzie, A., Wren, H., 1910. Experiments on the Walden inversion
part V. The interconversion of the optically active a-hydroxy-b-
phenylpropionic acids. J. Chem. Soc. 97, 1355–1358.
Ollagnier, S., Kervio, E., Retey, J., 1998. The role and source of 50-
deoxyadenosyl radical in a carbon skeleton rearrangement by a
plant enzyme. FEBS Lett. 437, 309–312.
O’Hagan, D., Robins, R.J., Wilson, M., Wong, C.W., Berry, M.,
Zabetakis, I., 1999. Fluorinated tropane alkaloids generated by
directed biosynthesis in transformed root cultures of Datura stra-
monium. J. Chem. Soc. Perkin Trans. 1, 2117–2120.
Patterson, S., O’Hagan, D., 2002. Isotopically labelled tropane alka-
loids. The synthesis of (RS)-[30,30-2H2]-and (RS)-[10-13C,30,30-2H2]-
hyoscyamines for metabolism studies in plants. J. Labelled Compd.
and Radiopharm. 45, 191–198.
Acknowledgements
Platt, R.V., Opie, C.T., Haslam, E., 1984. Plant proanthocyani-
dins 8. Biosynthesis of flavan-3-ols and other secondary plant-
products from (2S)-phenylalanine. Phytochemistry 23, 2211–
2217.
The EPSRC is acknowledged for providing a stu-
dentship (S.P.).
Robins, R.J., Parr, A.J., Bent, E.G., Rhodes, M.J.C., 1991. Studies on
the biosynthesis of tropane alkaloids in Datura stramonium L D15/
5. Transformed root cultures 1. The kinetics of alkaloid production
and the influence of feeding intermediate metabolites. Planta 183,
185–195.
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