N-methyltransferase activities in caffeine biosynthesis: Biochemical characterization and time course during leaf development of Coffea arabica

Abstract

In order to accurately measure and characterize the very labile methyltransferase activities catalysing the two last steps in caffeine biosynthesis (from 7-methylxanthine to theobromine, and from theobromine to caffeine by the 2nd and 3rd NMT activity, respectively), an assay was designed that renders the use of radioactivity superfluous. Since in the assay maximum velocity was achieved by sufficiently high concentrations of both the methyl donor and acceptor, sensitivity was equal to the conventional protocols using minute substance quantities of[methyl- 3H]- or[methyl- 14C]SAM of high specific radioactivity. For the above mentioned NMT activities of the coffee plant a maximum pH stability was found at pH 7.5. Temperature stability tests revealed an almost linear decrease by increasing temperature with the exception of the 3rd NMT that was relatively stable between 30 and 50°. The isoelectric point was located at pH 4.5 to 5.0 (2nd NMT), and the pH optima at 7 to 8 and 8.5 for the 2nd and 3rd NMT activity, respectively. The temperature optimum is broad and around 35° for both activities. Interestingly, the 2nd NMT activity has a lower K m for the methyl acceptor than the 3rd one, and this inversely for the methyl donor, SAM. Maximum relative and absolute methyltransferase activities coincide with leaf emergence, when, as expected, the chemical defence has highest priority and the purine alkaloids (PuA) are allocated at peak concentrations. The 2nd NMT activity was always considerably higher than the 3rd one. However, they frequently paralleled each other as they were components of one entity. The only transient accumulation of theobromine in the young leaflets suggestedly results from the competition of the two NMT activities for SAM and/or from a reduced supply of purine precursors in the preceding methylations.