Membrane vesicles from an Escherichia coli mutant with a deletion of the uncBC operon required ATP to translocate proteins, thus ruling out an essential role of F1F0-H+-ATPase in ATP-dependent protein translocation. Moreover, proteins could be translocated in the absence of proton motive force. At suboptimal ATP concentrations, D-lactate stimulated protein translocation, indicating that proton motive force, although insufficient to support translocation, could facilitate the process.The source of energy for protein translocation across bacterial membranes has been the focus of intensive studies. In intact bacteria, a requirement for an energized membrane was suggested by the observation that dissipation of the proton motive force (PMF) by a proton uncoupler or by valinomycin blocks processing and either insertion or secretion of several membrane and exported proteins (1,4,5,10,11,14). Moreover, PMF was found to be involved in protein translocation in vitro in a coupled translation-translocation system (13). However, it is not certain whether the effect of PMF on protein translocation is direct or secondary.With the finding that in vitro translocation can be accomplished posttranslationally (2, 8), it became possible to examine the energy requirements for protein translocation after removal by gel filtration of energy sources used for protein synthesis. (The translocated products were defined as materials [precursor and mature proteins] resistant to pronase digestion and to sedimenting with membrane vesicles.) We have found that translocation of both alkaline phosphatase and OmpA protein into Escherichia coli membrane vesicles requires ATP or an ATP-regenerating system rather than PMF alone (3). The efficiencies of translocation in this system were 10 to 25 and 25 to 35% for alkaline phosphatase and OmpA protein, respectively (2, 3). Moreover, ATP can still support protein translocation, though less efficiently, in the presence of proton uncouplers or with membranes prepared from mutants severely defective in the F1 fraction of the H+-ATPase (3), suggesting that neither PMF nor a functional H+-ATPase is essential for ATPdependent protein translocation. Nevertheless, PMF contributes to the optimal activity since proton uncouplers inhibit the activity by 50 to 70% (3).In the earlier experiments involving Fl-defective membranes, we used an S30 fraction prepared from E. coli D10 with normal H+-ATPase, which might have contained trace amounts of the functional F1 portion of the H+-ATPase in solubilized form capable of complementing the Fo portion of the Fl-defective membranes to provide ATP-dependent protein translocation. To eliminate this possibility and to determine whether the Fo portion of H+-ATPase might be involved in translocation (e.g., by providing a channel or pore), experiments were carried out with mutant CK1801 * Corresponding author.(F-AlacU169 araD139 thiA rpsL relA AuncBC), a derivative of MC4100 (obtained from C. Kumamoto of Stanford University) whose genes for both F1 and Fo of H+-ATPase are entirel...
Current clinical treatments for pneumococcal infections have many limitations and are faced with many challenges. New capsular polysaccharide structures must be explored to cope with diseases caused by different serotypes of Streptococcus pneumoniae. UDP-galactose 4-epimerase (GalE) is an essential enzyme involved in polysaccharide synthesis. It is an important virulence factor in many bacterial pathogens. In this study, we found that two genes (galEsp1 and galEsp2) are responsible for galactose metabolism in pathogenic S. pneumoniae TIGR4. Both GalESp1 and GalESp2 were shown to catalyze the epimerization of UDP-glucose (UDP-Glc)/UDP-galactose (UDP-Gal), but only GalESp2 was shown to catalyze the epimerization of UDP-N-acetylglucosamine (UDP-GlcNAc)/UDP-N-acetylgalactosamine (UDP-GalNAc). Interestingly, GalESp2 had 3-fold higher epimerase activity toward UDP-Glc/UDP-Gal than GalESp1. The biochemical properties of GalESp2 were studied. GalESp2 was stable over a wide range of temperatures, between 30 and 70°C, at pH 8.0. The K86G substitution caused GalESp2 to lose its epimerase activity toward UDP-Glc and UDP-Gal; however, substitution C300Y in GalESp2 resulted in only decreased activity toward UDP-GlcNAc and UDP-GalNAc. These results indicate that the Lys86 residue plays a critical role in the activity and substrate specificity of GalESp2.
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