Copalyl diphosphate (CPP) synthase from (PvCPS) is a bifunctional diterpene synthase with both prenyltransferase and class II cyclase activities. The prenyltransferase α domain catalyzes the condensation of C dimethylallyl diphosphate with three successively added C isopentenyl diphosphates (IPPs) to form C geranylgeranyl diphosphate (GGPP), which then undergoes a class II cyclization reaction at the βγ domain interface to generate CPP. The prenyltransferase α domain mediates oligomerization to form a 648-kD (αβγ) hexamer. In the current study, we explore prenyltransferase structure-function relationships in this oligomeric assembly-line platform with the goal of generating alternative linear isoprenoid... More
Copalyl diphosphate (CPP) synthase from (PvCPS) is a bifunctional diterpene synthase with both prenyltransferase and class II cyclase activities. The prenyltransferase α domain catalyzes the condensation of C dimethylallyl diphosphate with three successively added C isopentenyl diphosphates (IPPs) to form C geranylgeranyl diphosphate (GGPP), which then undergoes a class II cyclization reaction at the βγ domain interface to generate CPP. The prenyltransferase α domain mediates oligomerization to form a 648-kD (αβγ) hexamer. In the current study, we explore prenyltransferase structure-function relationships in this oligomeric assembly-line platform with the goal of generating alternative linear isoprenoid products. Specifically, we report steady-state enzyme kinetics, product analysis, and crystal structures of various site-specific variants of the prenyltransferase α domain. Crystal structures of the H786A, F760A, S723Y, S723F, and S723T variants have been determined at resolutions of 2.80, 3.10, 3.15, 2.65, and 2.00 Å, respectively. The substitution of S723 with bulky aromatic amino acids in the S723Y and S723F variants constricts the active site, thereby directing the formation of the shorter C isoprenoid, farnesyl diphosphate. While the S723T substitution only subtly alters enzyme kinetics and does not compromise GGPP biosynthesis, the crystal structure of this variant reveals a nonproductive binding mode for IPP that likely accounts for substrate inhibition at high concentrations. Finally, mutagenesis of the catalytic general acid in the class II cyclase domain, D313A, significantly compromises prenyltransferase activity. This result suggests molecular communication between the prenyltransferase and cyclase domains despite their distant connection by a flexible polypeptide linker.
