Abstracts Category : Other

Exploring the Monoterpene Cyclization Mechanism by Studying (+)-Limonene Synthase Using Novel Fluorinated Substrate Analogues

by Qi Yu

A proposed universal monoterpene synthases (cyclases) cyclization mechanism topologically requires an isomerization step of the substrate geranyl diphosphate (GPP) to the formation of the proposed universal intermediate linalyl diphosphate (LPP), which has never been directly observed. The study described here was designed to investigate the cyclization mechanism by studying a model enzyme, (4R)-(+)-limonene synthase ((+)-LS), a monoterpene cyclase that produces (+)-limonene by using substrate GPP. The study synthesized two novel difluorinated substrate analogues, 8,9-difluorogeranyl diphosphate (DFGPP) and 8,9-difluorolinalyl diphosphate (DFLPP), which were designed to retard the cyclization step in order to observe the intermediate at the enzyme active site. Both analogues are proved to be substrates for (+)-LS. Compared to GPP, which has a kcat value of 0.0905 +/- 0.0070 s-1, DFGPP has a reduced kcat value at 0.00144 +/- 0.00002 s-1, showing a 69-fold slower rate in the catalytic reaction. The KM value of DFGPP is 20.50 +/- 1.05 M, and does not change much compared to that of the nonfluorinated substrate with 29.79 +/- 7.83 M. Both DFGPP and DFLPP are catalyzed to make the same acyclic product and the structure of the product has been investigated through NMR. Our results indicate that LPP might be an intermediate in the ring closure mechanism for (+)-LS. Additionally, LPP is also proved to be a substrate for (+)-LS and makes (+)-limonene.