Purification and characterization of caffeine synthase from tea leaves.
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 microM, 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.
Kato
M
M
Department of Biology, Faculty of Science, Ochanomizu University, Tokyo 112-8610, Japan.
Mizuno
K
K
Fujimura
T
T
Iwama
M
M
Irie
M
M
Crozier
A
A
Ashihara
H
H
D000345:Affinity Labels; D000595:Amino Acid Sequence; D002110:Caffeine / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018515:Plant Leaves / Q000201:enzymology; D013379:Substrate Specificity; D013662:Tea / Q000201:enzymology* / Q000235:genetics
D000345:Affinity Labels; D013662:Tea; D002110:Caffeine; D008780:Methyltransferases; C120479:caffeine synthase