| Description |
Despite differing catalytic specificities and activities, farnesyl diphosphate synthase (FPPase) and chrysanthemyl diphosphate synthase (CPPase) from Artemisia tridentata ssp. spiciformis have sequence alignments showing 69% identity and 84% similarity. The active sites of the enzymes are formed by a six-membered, a-helical bundle representative of the type-I isoprenoid synthase fold (IS-1 fold). FPPase is selective for the 1'-4 coupling (chain elongation) of dimethylallyl diphosphate (DMAPP) and two isopentenyl diphosphate (IPP) molecules, initially forming geranyl diphosphate (GPP) and then farnesyl diphosphate (FPP). CPPase preferentially forms GPP when chain elongating, and can also couple two DMAPP molecules (irregular coupling) to preferentially form the c1'-1-2 product chysanthemyl diphosphate (CPP). CPPase additionally produces the 1'-2 product lavandulyl diphosphate (LPP) and a trace amount of the c1'-2-3-2' product maconelliyl diphosphate (MPP). The catalytic diversity of CPPase comes at the cost of catalytic efficiency (kcat/Km), as FPPase chain elongates at over 30,000-fold greater efficiency. In this study chimeric enzyme constructs were built from the IS-1 folds of FPPase and CPPase. Each enzyme was turned into the other through sequentially swapping the helices and loops of their IS-1 fold, building enzymes of varying FPPase and CPPase character to assess what structural elements affect catalytic specificity and activity. The first catalytic transformation observed along the N- to C-terminal conversion of FPPase to CPPase was a shift from preferential FPP to GPP formation. The GPPase showed over 2000-fold greater catalytic efficiency toward terminating chain elongation at the C10 product. Then, catalytic efficiency dropped to CPPase-like levels, correlating with a T194G FPPase to CPPase mutation of the KT motif in the fourth conserved region among E-chain elongation enzymes. Following, irregular terpenoid catalysis was observed in the form of preferential LPP formation, associated with F231Y and D235N mutations in the fifth conserved region. Preferential CPP production was dependent upon an enzyme having C-terminal sequence outside the IS-1 fold from CPPase. Replacement of the N-terminal region outside of the IS-1 fold of CPPase with FPPase sequence reclaimed GPP binding ability and FPP formation. A return to an FPPase-like catalytic efficiency was not observed in any chimera along the N- to C-terminal metamorphosis of CPPase to FPPase, further indicating the significance of the C-terminal region in the catalytic activity and specificity of the enzymes. |
