| Summary: | 博士 === 國立臺灣大學 === 生化科學研究所 === 95 === Over 23000 compounds constitute an important family of natural products named isoprenoids, which are built on the 5-carbon isopentenyl pyrophosphate (IPP). Isoprenyl pyrophosphate synthases (IPPs) involved in the biosynthesis of the linear isoprenoid polymers each catalyzes consecutive condensation reactions of a designated number of isopentenyl pyrophosphate (IPP) with a single farnesyl pyrophosphate (FPP). These enzymes are named trans- or cis-prenyltransferases according to the stereochemistry of the double bonds from IPP condensation.
Among trans-prenyltransferases, geranylgeranyl pyrophosphate synthase (GGPPs) catalyzes a condensation reaction of farnesyl pyrophosphate (FPP) with an isopentenyl pyrophosphate (IPP) to generate C20 geranylgeranyl pyrophosphate (GGPP), a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether linked lipid. In this study, we solved 6 crystal structures of S. cerevisiae GGPPs in complex with substrate FPP, with IPP and the substrate analog farnesyl thiopyrophosphate (FsPP), alternative substrate GPP, and product GGPP. Unlike in FPP synthase where the third Mg2+ ion coordinated by the second DDxxD motif participates in the substrate binding, only two Mg2+ ions are observed in S. cerevisiae GGPPs. IPP is bound in a positively-charged pocket with Arg39, His68, Arg85 and Tyr205. From the binding mode of the product GGPP, the pyrophosphate of GGPP binds to the IPP pyrophosphate binding site and the hydrocarbon moiety sits in the FPP hydrocarbon binding site. The binding mode for GGPP in yeast GGPPs structure is distinct from that of the product in the proposed inhibitory site of human GGPPs.
Bisphosphonate drugs used for osteoclast-mediated bone resorption and tumor-induced hypercalcemia are potent inhibitors of farnesyl pyrophosphate synthase (FPPs). Both FPP and GGPP are essential ligands for posttranslational modification of small GTPases such as Ras, Rac, and Rho for their biological functions. To design specific inhibitors for GGPPs, we have solved five S. cerevisiae GGPPs-bisphosphonates complex structures in this thesis (These bisphosphonates are kindly provided by Prof. Eric Oldfield). Unlike the previous finding that bisphosphonate inhibitors bind to the essential Mg2+ ions in the FPPs active site, these inhibitors also bind to GGPPs, but not necessary with Mg2+ ion. The bisphosphonates can bind to GGPPs in the FPP site, IPP site and/or the product feedback inhibitory site. Moreover, particular bisphosphonates with hydrophobic moiety bind to undecaprenyl pyrophosphate synthase, which is a cis-prenyltrasnferase and catalyzes chain elongation of FPP by condensation with 8 IPP to form C55 product as lipid carrier for bacterial peptidoglycan biosynthesis. Cis-prenytransferases share no sequence homology and structural similarity with the trans-enzymes. In each of the five structures investigated, we found that there were up to four binding sites per monomer. Three of the binding sites occupy the top of a “funnel” region, while the fourth site is situated at the bottom of the funnel. One inhibitor molecule occupies the FPP substrate binding site and none competes with IPP binding.
|
|---|
