Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit

Autor(es)

Schimpl, Flavia Camila ^[1] ; Kiyota, Eduardo ^[1] ; Sampaio Mayer, Juliana Lischka ^[2] ; de Carvalho Goncalves, Jose Francisco ^[3] ; da Silva, Jose Ferreira ^[4] ; Mazzafera, Paulo ^[1]

Afiliação do(s) autor(es)

^[1] Univ Estadual Campinas, Dept Biol Vegetal, IB, BR-13083970 Campinas, SP - Brazil

^[2] Univ Estadual Centro Oeste, Dept Biol, Guarapuava, PR - Brazil

^[3] Inst Nacl de Pesquisas da Amazonia, Manaus, AM - Brazil

^[4] Univ Fed Amazonas, Fac Ciencias Agr, Dept Prod Anim & Vegetal, Manaus, AM - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Phytochemistry; v. 105, p. 25-36, SEP 2014.
• Citações Web of Science: 16

Resumo

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. (C) 2014 Elsevier Ltd. All rights reserved. (AU)

• Processo FAPESP: 11/03266-6 - Análise da expressão do gene de cafeína sintase em frutos de guaraná (Paullina cupana var. sorbilis)
• Beneficiário: Flávia Camila Schimpl
• Modalidade de apoio: Bolsas no Brasil - Mestrado