Ah, man, it's a molecule delivered straight from the Gods.......
Plant Physiol 1999 Jun;120(2):579-86
Purification and characterization of caffeine synthase from tea leaves.
Kato M, Mizuno K, Fujimura T, Iwama M, Irie M, Crozier A, Ashihara H
Department of Biology, Faculty of Science, Ochanomizu University, Tokyo 112-8610, Japan (M.K., H.A.).
[Medline record in process]
Caffeine synthase (CS), the S-adenosylmethionine-dependent N-methyltransferase involved in the last two steps of caffeine
biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity
and a final specific activity of 5.7 nkat mg-1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange,
adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of
61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and
1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine,
and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no
7-N-methyltransferase activity toward xanthosine and xanthosine 5'-monophosphate. The Km values of CS for paraxanthine,
theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 &mgr;M, respectively. The possible
role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence
for CS showed little homology with other methyltransferases. |
