Probing the Catalytic Mechanisms of Cis-type and Trans-type Prenyltransferases Using the Synthesized Analogs of FPP and IPP

Geranylgeranyl pryophsophate synthase (GGPPs, C20), octaprenyl pyrophosphate synthase (OPPs, C40), and undecaprenyl pyrophosphate synthases (UPPs, C55) are prenyltransferases which catalyze chain elongation of farnesyl diphosphate (FPP) via 1’-4 condensation reaction with various numbers of isopentenyl pyrophosphate (IPP) units to generate different isoprenoids. Based on the stereochemistry of the double bond formed during IPP condensation, prenyltransferases are classified into trans- and cis-types. GGPPs and OPPs are trans-type prenyltransferase, while UPPs belongs to the cis-type. The possible mechanisms for 1’-4 condensation reaction that have been proposed are (1) ionization-condensation-elimination mechanism (sequential mechanism), and (2) condensation-elimination mechanism (concerted mechanism).
In this study, we synthesized analogs of FPP and IPP to probe the reaction mechanisms of these three prenyltransferases, particularly that of the cis-type UPPs. By substituting the methyl group at C3 position of IPP with an electron-withdrawing bromo group, the resulting analog of IPP, 3-bromo-3-butenyl diphosphate, slowed down the rate of the condensation reaction. Trapping of the farnesol intermediates in the catalytic active site of GGPPs and OPPs using radiolabeled FPP led us to propose that the reaction mechanisms of trans-type prenyltransferases go through an ionization- condensation-elimination mechanism. Using [1-14C] FPP as the substrate in place of [1-12C] FPP, the kinetic isotope effect on the consumption of IPP for UPPs reaction (1.14 ± 0.04 ) was smaller than those for OPPs reaction (1.61 ± 0.07) and GGPPs reaction (1.73 ± 0.07). The lack of apparent isotope effect and the fact that no farnesol intermediate was trapped, indicating that the cis-type UPPs reaction may proceed through a concerted condensation-elimination mechanism.