title: Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.
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 1083bp 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.
authors.affiliation: Departamento de Biologia Vegetal-IB, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil.
authors.forename: Flávia Camila
authors.initials: FC
authors.lastname: Schimpl
authors.affiliation: Departamento de Biologia Vegetal-IB, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Kiyota
authors.affiliation: Departamento de Biologia, Universidade Estadual do Centro-Oeste, Guarapuava, PR, Brazil.
authors.forename: Juliana Lischka Sampaio
authors.initials: JL
authors.lastname: Mayer
authors.affiliation: Instituto Nacional de Pesquisas da Amazônia, Manaus, AM, Brazil.
authors.forename: José Francisco de Carvalho
authors.initials: JF
authors.lastname: Gonçalves
authors.affiliation: Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, AM, Brazil.
authors.forename: José Ferreira
authors.initials: JF
authors.lastname: da Silva
authors.affiliation: Departamento de Biologia Vegetal-IB, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil. Electronic address: pmazza@unicamp.br.
authors.forename: Paulo
authors.initials: P
authors.lastname: Mazzafera
mesh_terms: D000470:Alkaloids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002110:Caffeine / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005638:Fruit / Q000737:chemistry; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D032126:Paullinia / Q000737:chemistry* / Q000235:genetics; D018515:Plant Leaves / Q000737:chemistry; D011687:Purines / Q000032:analysis / Q000737:chemistry; D012639:Seeds / Q000737:chemistry / Q000201:enzymology; D013805:Theobromine / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D013806:Theophylline / Q000032:analysis / Q000737:chemistry; D014970:Xanthines / Q000032:analysis / Q000737:chemistry
chemical_list: D000470:Alkaloids; D011687:Purines; D014970:Xanthines; D002110:Caffeine; D013806:Theophylline; C064273:7-methylxanthine; D008780:Methyltransferases; C120479:caffeine synthase; D013805:Theobromine; C030985:purine
keywords: Amazon; Caffeine; Paullinia cupana var. sorbilis; Recombinant protein; Sapindaceae; Seed; Theobromine; Venezuelan guarana