Biotechnology of Plant Secondary Metabolism

Flávia Camila Schimpl, Eduardo Kiyota, Paulo Mazzafera, Schimpl, Flávia Camila, Kiyota, Eduardo, Mazzafera, Paulo

Caffeine synthase (CS) is a methyltransferase responsible for the last two steps of the caffeine biosynthesis pathway in plants. CS is able to convert 7-methylxanthine to theobromine (3,7-dimethylxanthine) and theobromine to caffeine (1,3,7-trimethylxanthine) using S-adenosyl-L-methionine as the methyl donor in both reactions. The production of a recombinant protein is an important tool for the characterization of enzymes, particularly when the enzyme has affinity for different substrates. Guarana has the highest caffeine content among more than a hundred plant species that contain this alkaloid. Different from other plants, in which CS has a higher affinity for paraxanthine (1,7-dimethylxanthine), caffeine synthase from guarana (PcCS) has a higher affinity for theobromine. Here, we describe a method to produce a recombinant caffeine synthase from guarana in Escherichia coli and its purification by affinity chromatography. The recombinant protein retains activity and can be used in enzymatic assays and other biochemical characterization studies.