Cloning and Aggregation Characterization of Rubber Elongation Factor and Small Rubber Particle Protein from Ficus carica.

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are major latex proteins harvested from Hevea brasiliensis (the rubber tree; HbREF and HbSRPP, respectively). Their amino acid sequences exhibit high homology with each other. In the present study, we cloned two cDNAs encoding REF/SRPP-family proteins (FcREF/SRPP-1 and -2) from the laticifers of Ficus carica (fig tree). The amino acid sequences of these proteins showed high homology not only with each other but also with HbREF and HbSRPP. Recombinant FcREF/SRPP-1 and -2 were expressed in E. coli, and their aggregation properties were examined using a Congo red binding assay, agarose gel electrophoresis, and transmission electron microscopy. FcREF/SRPP-1 formed fibrils when incubated in PBS, and grew to micrometer-sized amorphous aggregates that precipitated rapidly. These aggregation properties of FcREF/SRPP-1 are quite similar to those of HbREF, although the growth rate and size of FcREF/SRPP-1 aggregates were inferior to those of HbREF. FcREF/SRPP-2 also formed aggregates during the incubation, but they did not precipitate, as has been reported for HbSRPP. Our results suggest that FcREF/SRPP-1 and -2 correspond to HbREF and HbSRPP, respectively. These aggregation properties could provide useful benchmarks for classifying REF/SRPP-family proteins as REF or SRPP.

Role of Cys73 in the thermostability of farnesyl diphosphate synthase from Geobacillus stearothermophilus.

Farnesyl diphosphate synthase (FPPase) is an enzyme that catalyzes the condensation between one molecule of dimethylallyl diphosphate (DMAPP) and two molecules of isopentenyl diphosphate (IPP) to produce farnesyl diphosphate (FPP). FPP is an important precursor in the isoprenoid synthesis pathway. In this study, the crystal structure of FPPase from Geobacillus stearothermophilus (GsFPPase) was determined at 2.31 Å resolution. The structure of GsFPPase shows a three-layered all α-helical fold and conserved functional domains similar to other prenyltransferases. We have analyzed the structural features of GsFPPase related to thermostability and compared it with those of human and avian mesophilic FPPases. "Semi-conserved" regions which appear to be possible features contributing to the thermostability of FPPase were found.

Synthesis of water-dispersible silver nanoparticles by thermal decomposition of water-soluble silver oxalate precursors.

Silver oxalate, one of the coordination polymer crystals, is a promising synthetic precursor for transformation into Ag nanoparticles without any reducing chemicals via thermal decomposition of the oxalate ions. However, its insoluble nature in solvents has been a great disadvantage, especially for systematic control of crystal growth of the Ag nanoparticles, while such control of inorganic nanoparticles has been generally performed using soluble precursors in homogeneous solutions. In this paper, we document our discovery of water-soluble species from the reaction between the insoluble silver oxalate and N,N-dimethyl-1,3-diaminopropane. The water-soluble species underwent low-temperature thermal decomposition of the oxalate ions at 30 °C with evolution of CO2 to reduce Ag+ to Ag0. Water-dispersible Ag nanoparticles have been successfully synthesized from the water-soluble species in the presence of gelatin via similar thermal decomposition at 100 °C. The gelatin-protected and water-dispersible Ag nanoparticles with a mean diameter of 25.1 nm appeared. In addition, antibacterial activity of the prepared water-dispersible Ag nanoparticles has been preliminarily investigated.

Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.

Class B ß-lactamases are known as metallo-ß-lactamases (MBLs) and they hydrolyze most ß-lactams, including carbapenems. IMP-18, an MBL cloned from Pseudomonas aeruginosa, was overexpressed, purified and crystallized by vapour diffusion for X-ray crystallographic analysis. Preliminary X-ray analysis showed that the crystal diffracted to 2.4 Å resolution and belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 120.77, c = 96.54 Å, α = ß = γ = 90°, suggesting the presence of two molecules in the asymmetric unit.

Kinetic studies of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase reaction using 3-desmethyl allylic substrate analogs.

In order to investigate the substrate binding feature of undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26 with respect to farnesyl diphosphate and a reaction intermediate, (Z,E,E)-geranylgeranyl diphosphate, we examined the reactivity of artificial substrate analogs, 3-desmethyl farnesyl diphosphate and 3-desmethyl Z-geranylgeranyl diphosphate, which lack the methyl group at the 3-position of farnesyl diphosphate and Z-geranylgeranyl diphosphate, respectively. Undecaprenyl diphosphate synthase did not accept either of the 3-desmethyl analogs as the allylic substrate, indicating that the methyl group at the 3-position of the allylic substrate is important in the undecaprenyl diphosphate synthase reaction. These analogs showed different inhibition patterns in the cis-prenyl chain elongation reaction with respect to the reactions of farnesyl diphosphate and Z-geranylgeranyl diphosphate as allylic substrate. These results suggest that the binding site for the natural substrate farnesyl diphosphate and those for the intermediate allylic diphosphate, which contains the cis-prenyl unit, are different during the cis-prenyl chain elongation reaction.

Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates.

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.