Purification and characterization of caffeine synthase from tea leaves

Plant Physiol. 1999 Jun;120(2):579-86. doi: 10.1104/pp.120.2.579.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels
  • Amino Acid Sequence
  • Caffeine / biosynthesis*
  • Electrophoresis, Polyacrylamide Gel
  • Hydrogen-Ion Concentration
  • Kinetics
  • Methyltransferases / genetics
  • Methyltransferases / isolation & purification*
  • Methyltransferases / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • Plant Leaves / enzymology
  • Substrate Specificity
  • Tea / enzymology*
  • Tea / genetics

Substances

  • Affinity Labels
  • Tea
  • Caffeine
  • Methyltransferases
  • caffeine synthase