rPfFPPS expressed as a GST fusion protein was used to characterize its functional activity and to determine the apparent kinetic parameters. Interestingly, the re moval of the GST tag from rPfFPPS resulted in almost complete activity loss. An active form of GGPPS from Thermus thermophilus and Sulfolobus acidocaldarius was also overexpressed in E. coli cells as a GST fusion protein. Ohto www.selleckchem.com/products/ganetespib-sta-9090.html et al. suggested that the presence of the GST tag leads to thermal stability of the recombinant enzymes. Previous studies have shown that many FPPS homo logues can Inhibitors,Modulators,Libraries accept both DMAPP and GPP as allylic sub strates. When synthesizing FPP from DMAPP, the enzyme catalyzes two condensation reactions with IPP, releasing only trace amounts of the intermediate GPP, while GGPPS can accept DMAPP, GPP, and FPP as substrates.
The Inhibitors,Modulators,Libraries activity of rPfFPPS and the parasite extracts were confirmed by purification of the synthesized products Inhibitors,Modulators,Libraries by RP HPLC. When DMAPP was used as a substrate, GPP was detected in minor amounts while FPP and GGPP were the predominant products. When the reaction was catalyzed with GPP as allylic substrate, the only products observed were FPP and GGPP. Accordingly, when FPP was used as sub strate, only GGPP was observed. No products were detected when GGPP was used as a substrate. Hence, rPfFPPS is a bifunctional FPPS GGPPS enzyme. Importantly, similar products were observed using a sec ond approach where alcoholic compounds were analysed by TLC. Finally, the structures of products GOH, FOH, and GGOH were confirmed by ESI MS MS.
The bifunctional property of rPfFPPS in producing GGPP as well as FPP was previously described only in three or ganisms, the archaebacterium M. thermoautotrophicum, Inhibitors,Modulators,Libraries maize, and T. gondii. A related enzyme was described Inhibitors,Modulators,Libraries by Artz et al. in Cryptosporidium parvum. Although this enzyme was annotated as an FPPS, it shows the capacity to produce GGPP and also longer polyisoprenes with the main prod ucts being C25 and C30 compounds with most of the substrates tested. This is indicative that the en zymes from P. falciparum and T. gondii have a rather limited product spectrum compared to the Crypto sporidium homologue. Amino acid sequence alignment of FPPS from differ ent organisms revealed conserved regions I to VII with two characteristic aspartate rich motifs, one in region II called FARM and in region VI called SARM. Wang and Ohnuma clearly demonstrated that the product chain lengths of natural FPPS and GGPPS are mainly regulated by the amino acid residues located at the fourth and fifth position upstream of the FARM region. These residues are at the bottom of the active site pocket, making direct interactions with the terminal region of the allylic products. For this reason, the site was designated selleck screening library the CLD re gion.