S-adenosyl-L- methionine: theobromine 1-N-methyl-transferase, an enzyme catalysing the synthesis of caffeine in coffee
This paper presents data on the purification and N-terminal sequence of S-adenosyl-L methionine: theobromine 1-N-methyltransferase (STM), the enzyme responsible for the methylation of theobromine leading to caffeine formation in coffee. STM was purified from developing endosperms of immature fruits by DEAE-cellulose, hydrophobic interaction and affinity chromatography, using S-adenosyl-L-homocysteine as a ligand. The enzyme showed an apparent Mr of ca 54 000 and ca 60 000 determined by gel filtration and SDS-PAGE, respectively. A pi of 5.1 was obtained by liquid chromatofocusing and 4.8 by isoelectrofocusing in polyacrylamide gel electrophoresis. Using theobromine as substrate, the K subíndice m value for S-adenosyl-L-methionine was 10 uM, being competitively inhibited by S-adenosyl-Lhomocysteine (K subíndice i=4.6 uM). STM is a bifunctional enzyme since it also methylated 7-methylxanthine, the immediate precursor of theobromine in the caffeine biosynthesis pathway. The specific activity of STM with 7- methylxanthine was ca 55 of that determined with theobromine. The K subíndice m values obtained for theobromine and 7- methylxanthine were 0.196 and 0.496 mM, respectively. STM was also purified from leaves using the same procedures used for endosperms, plus an additional chromatography on a Mono Q column; theobromine was used as substrate. Finally, the N-terminal sequence for the first 20 amino acids was obtained for STM purified from endosperms. No similarities were found with other methyltransferase sequences or other known proteins.
| Main Authors: | , , , |
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| Format: | biblioteca |
| Published: |
1994
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| Subjects: | COFFEA ARABICA, CAFE, CAFEINA, TEOBROMINA, ENZIMAS, METIONINA, ACTIVIDAD ENZIMATICA, |
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