Molecular and biochemical characterization of three aromatic polyketide synthase genes from Rubus idaeus

Three polyketide synthase genes (PKS1, PKS2, PKS3) from cell suspension cul tures of raspberry (Rubus idaeus L. cv. Royalty) were characterized. They s howed high similarity in both their nucleotide and deduced amino acid seque nces. All three proteins contain the amino acid residues identified in prev ious work as essential for chalcone synthase (CHS) function. Enzyme activit ies were investigated after heterologous expression in Escherichia coli. Ri PKS1 is a typical naringenin CHS that synthesizes the chalcone as the main reaction product, and p-coumaryltriacetic acid lactone (CTAL) as a minor by -product. RiPKS3 differed from RiPKS1 in four positions (K49R, M64R, P120L, V188A), and the products in vitro were predominantly CTAL and low levels o f chalcone. RiPKS2 had the same four differences from RiPKS1 as RiPKS3, but in addition two further exchanges (R259H, F344L), and the protein had no d etectable enzyme activity. Experiments with RiPKS1 containing either 259H o r 344L showed that each of the exchanges was sufficient to completely elimi nate enzyme activity. These experiments identify amino acid residues in CHS which are important for folding of the tetraketide intermediate to the cha lcone (PKS3) and which are in general essential for CHS activity (PKS2). Th e possible functions of these residues are discussed.