cDNA cloning, functional expression and characterization of ent-copalyl diphosphate synthase from Scoparia dulcis L.

Scoparia dulcis L. (Scrophulariaceae) produces several classes of diterpenes, which are proposed to be biosynthesized via syn-copalyl diphosphate (syn-CDP). On the other hand, gibberellin has been reported to be biosynthesized via ent-CDP and acts as a growth regulator. Therefore, S. dulcis should possess at least two enzymatic genes encoding ent- and syn-copalyl diphosphate synthases (CPSs) which catalyze cyclizations of GGDP. An RT-PCR method using degenerate primers based on the consensus sequences of plant CPSs allowed the isolation of a cDNA fragment showing a high similarity to plant CPSs. The full-length cDNA obtained using the rapid amplification of cDNA ends (RACE) method contained 2475 bp ORF encoding 825 amino acid residues. The recombinant protein expressed in Escherichia coli catalyzed cyclization of geranylgeranyl diphosphate (GGDP) to ent-CDP. Therefore, the enzyme was found to be ent-CPS, which we named SdECPS. The enzyme showed optimum activity at 30 °C and pH 7.4 and required Mg2+ ion as metallic co-factor. In addition, SdECPS was found to bear an N-terminal chloroplast targeting signal sequence and its truncated protein showed higher CPS activity than the native enzyme, while deletion of more than 111 amino acid residues of N-terminal sequence significantly reduced this activity.