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71.
Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two-dimensional NMR spectroscopy. 总被引:6,自引:5,他引:1 下载免费PDF全文
M. Wittekind P. Rajagopal B. R. Branchini J. Reizer M. H. Saier Jr R. E. Klevit 《Protein science : a publication of the Protein Society》1992,1(10):1363-1376
The solution structure of the phosphocarrier protein, HPr, from Bacillus subtilis has been determined by analysis of two-dimensional (2D) NMR spectra acquired for the unphosphorylated form of the protein. Inverse-detected 2D (1H-15N) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann-Hahn (HOHAHA) spectra utilizing 15N assignments (reported here) as well as previously published 1H assignments were used to identify cross-peaks that are not resolved in 2D homonuclear 1H spectra. Distance constraints derived from NOESY cross-peaks, hydrogen-bonding patterns derived from 1H-2H exchange experiments, and dihedral angle constraints derived from analysis of coupling constants were used for structure calculations using the variable target function algorithm, DIANA. The calculated models were refined by dynamical simulated annealing using the program X-PLOR. The resulting family of structures has a mean backbone rmsd of 0.63 A (N, C alpha, C', O atoms), excluding the segments containing residues 45-59 and 84-88. The structure is comprised of a four-stranded antiparallel beta-sheet with two antiparallel alpha-helices on one side of the sheet. The active-site His 15 residue serves as the N-cap of alpha-helix A, with its N delta 1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme I. The existence of a hydrogen bond between the side-chain oxygen atom of Tyr 37 and the amide proton of Ala 56 is suggested, which may account for the observed stabilization of the region that includes the beta-turn comprised of residues 37-40. If the beta alpha beta beta alpha beta (alpha) folding topology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of beta alpha beta beta alpha beta proteins that include acylphosphatase and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins. 相似文献
72.
Regulation of sugar uptake via the phosphoenolpyruvate-dependent phosphotransferase systems in Bacillus subtilis and Lactococcus lactis is mediated by ATP-dependent phosphorylation of seryl residue 46 in HPr. 总被引:1,自引:1,他引:0 下载免费PDF全文
By using both metabolizable and nonmetabolizable sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), we show that PTS sugar uptake into intact cells and membrane vesicles of Lactococcus lactis and Bacillus subtilis is strongly inhibited by high concentrations of any of several metabolizable PTS sugars. Inhibition requires phosphorylation of seryl residue 46 in the phosphocarrier protein of the PTS, HPr, by the metabolite-activated, ATP-dependent protein kinase. Inhibition does not occur when wild-type HPr is replaced by the S46A mutant form of this protein either in vesicles of L. lactis or B. subtilis or in intact cells of B. subtilis. Nonmetabolizable PTS sugar analogs such as 2-deoxyglucose inhibit PTS sugar uptake by a distinct mechanism that is independent of HPr(ser-P) and probably involves cellular phosphoenolpyruvate depletion. 相似文献
73.
Regulation of gluconeogenesis by the glucitol enzyme III of the phosphotransferase system in Escherichia coli. 总被引:3,自引:1,他引:2 下载免费PDF全文
The gut operon was subcloned into various plasmid vectors (M. Yamada and M. H. Saier, Jr., J. Bacteriol. 169:2990-2994, 1987). Constitutive expression of the plasmid-encoded operon prevented utilization of alanine and Krebs cycle intermediates when they were provided as sole sources of carbon for growth. Expression of the gutB gene alone (encoding the glucitol enzyme III), subcloned downstream from either the lactose promoter or the tetracycline resistance promoter, inhibited utilization of the same compounds. On the other hand, overexpression of the gutA gene (encoding the glucitol enzyme II) inhibited the utilization of a variety of sugars as well as alanine and Krebs cycle intermediates by an apparently distinct mechanism. Phosphoenolpyruvate carboxykinase activity was greatly reduced in cells expressing high levels of the cloned gutB gene but was nearly normal in cells expressing high levels of the gutA gene. A chromosomal mutation in the gutR gene, which gave rise to constitutive expression of the chromosomal gut operon, also gave rise to growth inhibition on gluconeogenic substrates as well as reduced phosphoenolpyruvate carboxykinase activity. Phosphoenolpyruvate synthase activity in general varied in parallel with that of phosphoenolpyruvate carboxykinase. These results suggest that high-level expression of the glucitol enzyme III of the phosphotransferase system can negatively regulate gluconeogenesis by repression or inhibition of the two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase. 相似文献
74.
Genetic dissection of catalytic activities of the Salmonella typhimurium mannitol enzyme II. 总被引:7,自引:3,他引:4 下载免费PDF全文
Approximately 60 mutants of Salmonella typhimurium were isolated which exhibited altered levels of the activities of the mannitol enzyme II. The mutants were grouped into six distinct categories based on their mannitol fermentation, transport, chemotaxis, and phosphorylation activities. 相似文献
75.
Regulation of methyl-beta-d-thiogalactopyranoside-6-phosphate accumulation in Streptococcus lactis by exclusion and expulsion mechanisms 总被引:30,自引:25,他引:5 下载免费PDF全文
Starved cells of Streptococcus lactis ML3 (grown previously on galactose, lactose, or maltose) accumulated methyl-beta-D-thiogalactopyranoside (TMG) by the lactose:phosphotransferase system. More than 98% of accumulated sugar was present as a phosphorylated derivative, TMG-6-phosphate (TMG-6P). When a phosphotransferase system sugar (glucose, mannose, 2-deoxyglucose, or lactose) was added to the medium simultaneously with TMG, the beta-galactoside was excluded from the cells. Galactose enhanced the accumulation of TMG-6P. Glucose, mannose, lactose, or maltose plus arginine, was added to a suspension of TMG-6P-loaded cells of S. lactis ML3, elicited rapid expulsion of intracellular solute. The material recovered in the medium was exclusively free TMG. Expulsion of galactoside required both entry and metabolism of an appropriate sugar, and intracellular dephosphorylation of TMG-6P preceded efflux of TMG. The rate of dephosphorylation of TMG-6P by permeabilized cells was increased two-to threefold by adenosine 5'-triphosphate but was strongly inhibited by fluoride. S. lactis ML3 (DGr) was derived from S. lactis ML3 by positive selection for resistance to 2-deoxy-D-glucose and was defective in the enzyme IIMan component of the glucose:phosphotransferase system. Neither glucose nor mannose excluded TMG from cells of S. lactic ML3 (DGr), and these two sugars failed to elicit TMG expulsion from preloaded cells of the mutant strain. Accumulation of TMG-6P by S. lactis ML3 can be regulation by two independent mechanisms whose activities promote exclusion or expulsion of galactoside from the cell. 相似文献
76.
G Kapadia C C Chen P Reddy M H Saier J Reizer O Herzberg 《Journal of molecular biology》1991,221(4):1079-1080
The IIA domain of the glucose permease of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) from Bacillus subtilis has been crystallized. Crystals are obtained from ammonium sulfate solution. They diffract to at least 2.2 A resolution, and belong to space group C222(1), with unit cell dimensions: a = 74.2 A; b = 54.9 A; c = 67.0 A. 相似文献
77.
Evolution of the MIP family of integral membrane transport proteins 总被引:17,自引:0,他引:17
G. M. Pao L.-F. Wu K. D. Johnson H. Höfte M. J. Chrispeels G. Sweet N. N. Sandal M. H. Saier Jr 《Molecular microbiology》1991,5(1):33-37
Six integral membrane proteins of bacterial, animal, and plant origin, which are believed to function in solute transport, share sequence identity and are grouped together as members of the MIP family. These include the Escherichia coli glycerol facilitator, the major intrinsic protein from bovine lens fibre junction membranes, a plant tonoplast membrane protein, a soybean protein from the peribacteroid membrane, and a Drosophila neurogenic protein. These proteins, each of which appears to consist of six transmembrane helical segments per subunit, apparently arose by internal duplication of a three-transmembrane segment. Phylogenetic‘trees’interrelating these proteins and segments are presented. 相似文献
78.
79.
Purification and properties of D-mannitol-1-phosphate dehydrogenase and D-glucitol-6-phosphate dehydrogenase from Escherichia coli. 总被引:8,自引:6,他引:2 下载免费PDF全文
D-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and D-glucitol-6-phosphate dehydrogenase (EC 1.1.1.140) were purified to apparent homogeneity in good yields from Escherichia coli. The amino acid compositions, N-terminal amino acid sequences, sensitivities to chemical reagents, and catalytic properties of the two enzymes were determined. Both enzymes showed absolute specificities for their substrates. The subunit molecular weights of mannitol-1-phosphate and glucitol-6-phosphate dehydrogenases were 40,000 and 26,000, respectively; the apparent molecular weights of the native proteins, determined by gel filtration, were 40,000 and 117,000, respectively. It is therefore concluded that whereas mannitol-1-phosphate dehydrogenase is a monomer, glucitol-6-phosphate dehydrogenase is probably a tetramer. These two proteins differed in several fundamental respects. 相似文献
80.
Statistical and functional analyses of viral and cellular proteins with N-terminal amphipathic alpha-helices with large hydrophobic moments: importance to macromolecular recognition and organelle targeting. 总被引:6,自引:1,他引:5 下载免费PDF全文
A total of 1,911 proteins with N-terminal methionyl residues were computer screened for potential N-terminal alpha-helices with strong amphipathic character. By the criteria of D. Eisenberg (Annu. Rev. Biochem. 53:595-623, 1984), only 3.5% of nonplastid, nonviral proteins exhibited potential N-terminal alpha-helices, 18 residues in length, with hydrophobic moment values per amino acyl residue ([muH]) in excess of 0.4. By contrast, 10% of viral proteins exhibited corresponding [muH] values in excess of 0.4. Of these viral proteins with known functions, 55% were found to interact functionally with nucleic acids, 30% were membrane-interacting proteins or their precursors, and 15% were structural proteins, primarily concerned with host cell interactions. These observations suggest that N-terminal amphipathic alpha-helices of viral proteins may (i) function in nucleic acid binding, (ii) facilitate membrane insertion, and (iii) promote host cell interactions. Analyses of potential amphipathic N-terminal alpha-helices of cellular proteins are also reported, and their significance to organellar or envelope targeting is discussed. 相似文献