Connection of transport and sensing by UhpC, the sensor for external glucose-6-phosphate in Escherichia coli.

UhpC is a membrane-bound sensor protein in Escherichia coli required for recognizing external glucose-6-phosphate (Glc6P) and induction of the transport protein UhpT. Recently, it was shown that UhpC is also able to transport Glc6P. In this study we investigated whether these transport and sensing activities are obligatorily coupled in UhpC. We expressed a His-UhpC protein in a UhpC-deficient E. coli strain and verified that this construct does not alter the basic biochemical properties of the Glc6P sensor system. The effects of arginine replacements, mutations of the central loop, and introduction of a salt bridge in UhpC on transport and sensing were compared. The exchanges R46C, R266C and R149C moderately affected transport by UhpC but strongly decreased the sensing ability. This suggested that the affinity for Glc6P as a transported substrate is uncoupled in UhpC from its affinity for Glc6P as an inducer. Four of the 11 arginine mutants showed a constitutive phenotype but had near wild-type transport activity suggesting that Glc6P can be transported by a molecule locked in the inducing conformation. Introduction of an intrahelical salt bridge increased the transport activity of UhpC but abolished sensing. Three conserved residues from the central loop were mutated and although none of these showed transport, one exhibited increased affinity for sensing. Taken together, these data show that transport by UhpC is not required for sensing, that conserved arginine residues are important for sensing and not for transport, and that residues located in the central hydrophilic loop are critical for transport and for sensing.     

Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase

Glucose is metabolized in Escherichia coli chiefly via the phosphoglucose isomerase reaction; mutants lacking that enzyme grow slowly on glucose by using the hexose monophosphate shunt. When such a strain is further mutated so as to yield strains unable to grow at all on glucose or on glucose-6-phosphate, the secondary strains are found to lack also activity of glucose-6-phosphate dehydrogenase. The double mutants can be transduced back to glucose positivity; one class of transductants has normal phosphoglucose isomerase activity but no glucose-6-phosphate dehydrogenase. An analogous scheme has been used to select mutants lacking gluconate-6-phosphate dehydrogenase. Here the primary mutant lacks gluconate-6-phosphate dehydrase (an enzyme of the Enter-Doudoroff pathway) and grows slowly on gluconate; gluconate-negative mutants are selected from it. These mutants, lacking the nicotinamide dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase, grow on glucose at rates similar to the wild type. Thus, these enzymes are not essential for glucose metabolism in E. coli.     

Functional expression of human glucose-6-phosphate dehydrogenase in Escherichia coli

The complete coding sequence for human glucose-6-phosphate-dehydrogenase (G6PD) was inserted downstream from the tac promoter of a plasmid, pJF118EH, which also carries the lacIq repressor gene. When Escherichia coli strains (that are unable to grow on glucose due to the absence of functional zwf (G6PD-) and pgi genes) were transformed with this plasmid (pAC1), they were able to grow on glucose as sole carbon source. The rate of growth on glucose was faster in the presence of the inducer of the tac promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG). Extracts of the transformed cells contained a G6PD activity that was not detectable in the parental strains and that was inducible by IPTG. The G6PD activities from normal E. coli and from pAC1-transformed cells comigrated with human G6PD when subjected to electrophoresis on agarose gels. However, when denatured, the G6PD produced by pAC1 was, like the human enzyme, distinguishable from the E. coli-encoded enzyme on the basis of its immunoreactivity with antibody specific for human G6PD. Therefore, human G6PD can be expressed in E. coli and can function to complement the bacterial enzyme deficiency.     

Selective neutrality of glucose-6-phosphate dehydrogenase allozymes in Escherichia coli

Six naturally occurring alleles representing four electromorphs of the enzyme glucose-6-phosphate dehydrogenase were transferred by P1- mediated transduction from natural isolates of Escherichia coli into the genetic background of E. coli K12 and were studied in pairwise competition in chemostats limited for glucose in order to estimate differences in growth rate associated with the alleles. Although the level of resolution of such experiments is a growth rate differential of approximately 0.002 h-1, no significant differences among the strains were found. Studies of apparent Km and Vmax in crude enzyme extracts of the strains also failed to reveal any significant differences among the electromorphs. These results support the view that the alleles are selectively neutral or nearly neutral under these conditions.      

The signature motif in human glucose-6-phosphate transporter is essential for microsomal transport of glucose-6-phosphate

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT). Sequence alignments identify a signature motif shared by G6PT and a family of transporters of phosphorylated metabolites. Two null signature motif mutations have been identified in the G6PT gene of GSD-Ib patients. In this study, we characterize the activity of seven additional mutants within the motif. Five mutants lack microsomal G6P uptake activity and one retains residual activity, suggesting that in G6PT the signature motif is a functional element required for microsomal glucose-6-phosphate transport.     

Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts

The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.     

Expression of both Chlamydia pneumoniae RNase HIIs in Escherichia coli

Both genes encoding the RNase HIIs from Chlamydia pneumoniae AR 39 (discriminated as CpRNase HIIa and CpRNase HIIb in this report) were cloned and efficiently expressed in Escherichia coli. These genes amplified from Chlamydial genomes with PCR were digested with restriction endonucleases and then cloned into plasmid pET-28a predigested with the same enzymes. DNA sequencing confirmed that the constructs were correct in translation frame and coding sequence. Recombinant RNase HIIs were over-expressed by 0.5 mM IPTG induction. CpRNase HIIa existed mainly as inclusion bodies while CpRNase HIIb mainly as soluble fractions in E. coli. The soluble proteins were 20% of total expressed CpRNase HIIa and 65% of total expressed CpRNase HIIb, respectively. Native purification and denaturing Ni-NTA purification were performed to recover the recombinant CpRNase HIIs from induced bacteria. 3.36 mg CpRNase HIIa and 18 mg CpRNase HIIb were, respectively, obtained from 1 g wet bacteria with native Ni-NTA purification. Denaturing Ni-NTA purification recovered 14.48 mg CpRNase HIIa and 10.4 mg CpRNase HIIb from 1 g wet bacteria, respectively. Although the proteins recovered by denaturing Ni-NTA purification were inactive, re-folding by dialysis against decreased concentrations of urea could generate CpRNase HIIa and CpRNase HIIb as active as those recovered by native Ni-NTA purification. These efforts offered basis for further study on the structure-function relationships and their biological importance of Chlamydial RNase HIIs.     

Isomerase activity of the C-terminal fructose-6-phosphate binding domain of glucosamine-6-phosphate synthase from Escherichia coli

The isomerase activity of the C-terminal fructose-6P binding domain (residues 241-608) of glucosamine-6-phosphate synthase from Escherichia coli has been studied. The equilibrium constant of the C-terminal domain k(eq) ([glucose-6P]/[fructose-6-P]) = 5.0. A non-competitive product inhibition of the isomerase activity by the reaction product glucose-6-P has been detected. The existence of more than one binding and reaction sites for the substrate fructose-6P on the molecule of glucosamine-6-phosphate synthase can be expected. The fructose-6P binding domain possibly includes a regulatory site, different from the catalytic center of the enzyme.     

Crystal structure of Escherichia coli glucose-1-phosphate thymidylyltransferase (RffH) complexed with dTTP and Mg2+

The enzyme glucose-1-phosphate thymidylyltransferase (RffH), the product of the rffh gene, catalyzes one of the steps in the synthesis of enterobacterial common antigen (ECA), a cell surface glycolipid found in Gram-negative enteric bacteria. In Escherichia coli two gene products, RffH and RmlA, catalyze the same enzymatic reaction and are homologous in sequence; however, they are part of different operons and function in different pathways. We report the crystal structure of RffH bound to deoxythymidine triphosphate (dTTP), the phosphate donor, and Mg(2+), refined at 2.6 A to an R-factor of 22.3% (R(free) = 28.4%). The crystal structure of RffH shows a tetrameric enzyme best described as a dimer of dimers. Each monomer has an overall alpha/beta fold and consists of two domains, a larger nucleotide binding domain (residues 1-115, 222-291) and a smaller sugar-binding domain (116-221), with the active site located at the domain interface. The Mg(2+) ion is coordinated by two conserved aspartates and the alpha-phosphate of deoxythymidine triphosphate. Its location corresponds well to that in a structurally similar domain of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). Analysis of the RffH, RmlA, and GlmU complexes with substrates and products provides an explanation for their different affinities for Mg(2+) and leads to a proposal for the dynamics along the reaction pathway.     

Sulfate-mediated affinity chromatography on NADP+-Sepharose of glutamate dehydrogenase from halophilic bacteria and of glucose-6-phosphate dehydrogenase from Escherichia coli.

An improved synthesis of the 8-(6-aminohexyl)amino derivative of NADP+ is described for use in affinity chromatography. The binding of glutamate dehydrogenase isolated from halobacterium of the Dead Sea on a column of Sepharose linked to this NADP+ derivative could be drastically enhanced by addition of sulfate (1M) and provided a tool for partially purifying the enzyme from a crude extract. A similar finding is reported for glucose-6-phosphate dehydrogenase in crude extracts of Escherichia coli. The effects are shown to be biospecific, suggesting that the strength of the interaction between protein and immobilized coenzymes is a function of the sulfate concentration.     

The purification and properties of N-acetylglucosamine 6-phosphate deacetylase from Escherichia coli

1. N-Acetylglucosamine 6-phosphate deacetylase and 2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10, glucosamine 6-phosphate deaminase) of Escherichia coliK(12) have been separated by chromatography on DEAE-cellulose. 2. N-Acetylglucosamine 6-phosphate deacetylase has optimum pH8.5 and K(m) 0.8mm. Glucosamine 6-phosphate is a product of the reaction. There appear to be no essential cofactors. Glucosamine 6-phosphate and fructose 6-phosphate inhibit deacetylation. 3. Glucosamine 6-phosphate deaminase has optimum pH7.0 and K(m) 9.0mm. It is stimulated by N-acetylglucosamine 6-phosphate. 4. We propose that the deacetylase be termed 2-acetamido-2-deoxy-d-glucose 6-phosphate amidohydrolase (EC 3.5.1.-), with acetylglucosamine 6-phosphate deacetylase as a trivial name.     

Structural analysis of N-acetylglucosamine-6-phosphate deacetylase apoenzyme from Escherichia coli

We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis.