Topographical characterization of the ubiquinone reduction site of glucose dehydrogenase in Escherichia coli using depth-dependent fluorescent inhibitors.
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Membrane-bound glucose dehydrogenase in Escherichia coli possesses a binding site for ubiquinone as well as glucose, metal ion and pyrroloquinoline quinone. To probe the depth of the ubiquinone binding site in the membrane environment, we synthesized two types of fluorenyl fatty acids which bear an inhibitor mimic moiety (i.e., specific inhibitor capsaicin) close to the fluorene located at different positions in the alkyl tail chain; one close to the polar carbonyl head group (alpha-(3, 4-dimethoxyphenyl)acetyloxy-7-nonyl-2-fluoreneacetic acid, alpha-DFA), and the other in the middle of the chain (theta-(3, 4-dimethoxyphenyl)acetyloxy-7-ethyl-2-fluorenenonanoic acid, theta-DFA). Mixed lipid vesicles consisting of phosphatidylcholine (PC) and alpha-DFA or theta-DFA were prepared by sonication method, and fluorescent quenching against a hydrophilic quencher, iodide anion, was examined. The vesicles containing alpha-DFA were more susceptible to quenching than those containing theta-DFA, indicating that the fluorene and consequently capsaicin mimic moiety are located at different depths in the lipid bilayer depending upon the position of attachment to the alkyl tail chain. The purified glucose dehydrogenase was reconstituted into PC vesicles which consisted of PC and alpha-DFA or theta-DFA with various molar ratios. For both types of reconstituted vesicles, the extent of inhibition of short-chain ubiquinone reduction activity increased with increases in the molar ratio of fluorenyl fatty acid to PC. The ubiquinone reduction activity was more significantly inhibited in the reconstituted vesicles containing alpha-DFA compared to those containing theta-DFA. Our findings strongly suggested that the ubiquinone reduction site in glucose dehydrogenase is located close to the membrane surface rather than in the hydrophobic membrane interior. (+info)
Identification of a gene in Staphylococcus xylosus encoding a novel glucose uptake protein.
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By transposon Tn917 mutagenesis, two mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild type. Both transposons integrated in a gene, designated glcU, encoding a protein involved in glucose uptake in S. xylosus, which is followed by a glucose dehydrogenase gene (gdh). Glucose-mediated repression of beta-galactosidase, alpha-glucosidase, and beta-glucuronidase activities was partially relieved in the mutant strains, while repression by sucrose or fructose remained as strong as in the wild type. In addition to the pleiotropic regulatory effect, integration of the transposons into glcU reduced glucose dehydrogenase activity, suggesting cotranscription of glcU and gdh. Insertional inactivation of the gdh gene and deletion of the glcU gene without affecting gdh expression showed that loss of GlcU function is exclusively responsible for the regulatory defect. Reduced glucose repression is most likely the consequence of impaired glucose uptake in the glcU mutant strains. With cloned glcU, an Escherichia coli mutant deficient in glucose transport could grow with glucose as sole carbon source, provided a functional glucose kinase was present. Therefore, glucose is internalized by glcU in nonphosphorylated form. A gene from Bacillus subtilis, ycxE, that is homologous to glcU, could substitute for glcU in the E. coli glucose growth experiments and restored glucose repression in the S. xylosus glcU mutants. Three more proteins with high levels of similarity to GlcU and YcxE are currently in the databases. It appears that these proteins constitute a novel family whose members are involved in bacterial transport processes. GlcU and YcxE are the first examples whose specificity, glucose, has been determined. (+info)
A spectroscopic assay for the analysis of carbohydrate transport reactions.
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A carbohydrate-transport assay was developed that does not require isotopically labelled substrates, but allows transport reactions to be followed spectrophotometrically. It makes use of a membrane system (hybrid membranes or proteoliposomes) bearing the transport system of interest, and a pyrroloquinoline quinone-dependent aldose dehydrogenase [soluble glucose dehydrogenase (sGDH)] and the electron acceptor 2,6-dichloroindophenol (Cl2Ind) enclosed in the vesicle lumen. After transport across the vesicular membrane, the sugar is oxidized by sGDH. The accompanying reduction of Cl2Ind results in a decrease in A600. The assay was developed and optimized for the lactose carrier (LacS) of Streptococcus thermophilus, and both solute/H+ symport and exchange types of transport could be measured with high sensitivity in crude membranes as well as in proteoliposomes. To observe exchange transport, the membranes were preloaded with a nonoxidizable substrate analogue and diluted in assay buffer containing an oxidizable sugar. Transport rates measured with this assay are comparable with those obtained with the conventional assay using isotopically labelled substrates. The method is particularly suited for determining transport reactions that are not coupled to any form of metabolic energy such as uniport reactions, or for characterizing mutant proteins with a defective energy-coupling mechanism or systems with high-affinity constants for sugars. (+info)
A superoxide dismutase from the archaeon Sulfolobus solfataricus is an extracellular enzyme and prevents the deactivation by superoxide of cell-bound proteins.
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An oxygen-induced iron superoxide dismutase was found in the culture fluid of the thermoacidophilic crenarchaeon Sulfolobus solfataricus during growth on glucose-rich media. This protein was also identified as being associated with the cell-surface, with the amount of the released and cell-bound protein fractions depending on the growth phase of the cells. The steady decrease in cell-associated superoxide dismutase during continued growth correlated with the increase of free superoxide dismutase in the medium. Both enzyme fractions were purified to homogeneity and found to be active with different catalytic efficiency, with the released superoxide dismutase showing a fourfold lower specific activity. Characterization in comparison with the cytosolic superoxide dismutase revealed identical N-terminal sequences, electrophoretic mobility, isoelectric point, and molecular mass for all three differently located enzymes. In order to clarify the physiological role of the cell-associated superoxide dismutase, the prevention of cell-bound protein deactivation by oxyradicals was also investigated. Glucose dehydrogenase, which was chosen as a model enzyme, was demonstrated to be located on the cell surface and to be inactivated by potassium superoxide by in vivo assays. The direct protective effect of superoxide dismutase on glucose dehydrogenase was demonstrated by in vitro assays on the free released enzyme. Similarly, the prevention of deactivation by potassium superoxide was also demonstrated for the integral membrane protein succinate dehydrogenase by intact cell assay. Superoxide dismutase added to cells was shown to moderately reduce the critical damaging peroxidation and hence play a major role in maintaining the integrity of the outer cell envelope components. (+info)
Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase.
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Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2). W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2). (+info)
Aggregation, dissociation and unfolding of glucose dehydrogenase during urea denaturation.
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The effect of urea on glucose dehydrogenase from Bacillus megaterium has been studied by following changes in enzymatic activity, conformation and state of aggregation. It was found that the denaturation process involves several transitions. At very low urea concentrations (below 0.5 M), where the enzyme is fully active and tetrameric, there is a conformational change as monitored by an increase in intensity of the tryptophan fluorescence and a maximum exposure of organized hydrophobic surfaces as reported by the fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5.5'-disulfonic acid. At slightly higher urea concentrations (0.75-2 M), a major conformational transition occurs, as monitored by circular dichroism and fluorescence measurements, in which the enzyme activity is completely lost and is concomitant with the formation of interacting intermediates that lead to a highly aggregated state. Increasing urea concentrations cause a complete dissociation to lead first a partially and eventually the complete unfolded monomer. These phenomena are fully reversible by dilution of denaturant. It is concluded that after urea denaturation, the folding/assembly pathway of glucose dehydrogenase occurs with the formation of intermediate species in which transient higher aggregates appear to be involved. (+info)
Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation.
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A major structural element of bacterial endospores is a peptidoglycan (PG) wall. This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers. The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination. The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration, and is degraded during spore germination. Theories suggest that the spore PG may also play a mechanical role in the attainment of spore dehydration. Inherent in one of these models is the production of a gradient of cross-linking across the span of the spore PG. We report analyses of the structure of PG found within immature, developing Bacillus subtilis forespores. The germ cell wall PG is synthesized first, followed by the cortex PG. The germ cell wall is relatively highly cross-linked. The degree of PG cross-linking drops rapidly during synthesis of the first layers of cortex PG and then increases two- to eightfold across the span of the outer 70% of the cortex. Analyses of forespore PG synthesis in mutant strains reveal that some strains that lack this gradient of cross-linking are able to achieve normal spore core dehydration. We conclude that spore PG with cross-linking within a broad range is able to maintain, and possibly to participate in, spore core dehydration. Our data indicate that the degree of spore PG cross-linking may have a more direct impact on the rate of spore germination and outgrowth. (+info)
Structural requirements of pyrroloquinoline quinone dependent enzymatic reactions.
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On the basis of crystal structures of the pyrroloquinoline quinone (PQQ) dependent enzymes methanol dehydrogenase (MDH) and soluble glucose dehydrogenase (s-GDH), different catalytic mechanisms have been proposed. However, several lines of biochemical and kinetic evidence are strikingly similar for both enzymes. To resolve this discrepancy, we have compared the structures of these enzymes in complex with their natural substrates in an attempt to bring them in line with a single reaction mechanism. In both proteins, PQQ is located in the center of the molecule near the axis of pseudo-symmetry. In spite of the absence of significant sequence homology, the overall binding of PQQ in the respective active sites is similar. Hydrogen bonding interactions are made with polar protein side chains in the plane of the cofactor, whereas hydrophobic stacking interactions are important below and above PQQ. One Arg side chain and one calcium ion are ligated to the ortho-quinone group of PQQ in an identical fashion in either active site, in agreement with their proposed catalytic function of polarizing the PQQ C5-O5 bond. The substrates are bound in a similar position above PQQ and within hydrogen bond distance of the putative general bases Asp297 (MDH) and His144 (s-GDH). On the basis of these similarities, we propose that MDH and s-GDH react with their substrates through an identical mechanism, comprising general base-catalyzed hydride transfer from the substrate to PQQ and subsequent tautomerization of the PQQ intermediate to reduced PQQ. (+info)