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Title: | Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit |
Authors: | Schimpl, Flávia Camila Kiyota, Eduardo Mayer, Juliana Lischka Sampaio Gonçalves, José Francisco de Carvalho Silva, José Ferreira da Mazzafera, Paulo |
Keywords: | Paullinia Cupana Sapindaceae Theobroma Cacao 7-methylxanthine Alkaloid Caffeine Caffeine Synthase Methyltransferase Purine Purine Derivative Theobromine Theophylline Xanthine Derivative Amino Acid Sequence Chemical Structure Chemistry Enzymology Fruit Genetics Isolation And Purification Metabolism Methylation Nucleotide Sequence Paullinia Plant Leaf Seed Plant Alkaloids Amino Acid Sequence Base Sequence Caffeine Fruit Methylation Methyltransferases Molecular Structure Paullinia Plant Leaves Purines Seeds Theobromine Theophylline Xanthines |
Issue Date: | 2014 |
metadata.dc.publisher.journal: | Phytochemistry |
metadata.dc.relation.ispartof: | Volume 105, Pags. 25-36 |
Abstract: | Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083 bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein. © 2014 Elsevier Ltd. All rights reserved. |
metadata.dc.identifier.doi: | 10.1016/j.phytochem.2014.04.018 |
Appears in Collections: | Artigos |
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