Immunochemical comparison of phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase among the Enterobacteriaceae.

The bifunctional enzyme of the tryptophan operon, phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase (PRAI-InGPS;EC 4.1.1.48), was characterized by an immunochemical study of six representative members of the Enterobacteriaceae: Escherichia coli, Salmonella typhimurium, Enterobacter aerogenes, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris. PRAI-InGPS was purified from E. coli, and antisera were prepared in rabbits. These antisera were utilized in quantitative microcomplement fixation allowing for a comparison of the overall antigenic surface structure of the various homologous enzymes. These data showed E. coli PRAI-InGPS and S. marcescens and E. carotovora PRAI-InGPS (taken as a group) to have an index of dissimilarity of approximately 10, whereas the other organisms had values intermediate. In addition, antiserum to E. coli tryptophan synthetase beta2 subunit was used in microcomplement fixation to extend the previous comparison of this subunit (Rocha, Crawford, and Mills, 1972) to E. carotovora and P. vulgaris. Indexes of dissimilarity for E. coli compared to P. vulgaris of E. carotovora were 1.0 and 1.7, respectively. Agar immunodiffusion using PRAI-Ingps antisera showed significant cross-reaction among E. coli, E. aerogenes, S. typhimurium, and P. vulgaris whereas the enzymes from S. marcescens and E. carotovora cross-reacted to a lesser extent, with the latter reaction being quite weak. Comparative enzyme neutralization using E. coli PRAI-InGPS antisera showed significant cross-reactions among the enzymes in that all were neutralized at least 25%. The data taken together indicate that the trpC gene products in the Enterobacteriaceae are a homologous group of proteins, that the genetic divergene of the trpC gene is basically the same as the trpA gene, and that both are less conserved than the trpB gene. Furthermore, the PRAI-InGPS, enzyme active site appears to represent a more evolutionarily conserved region of the protein. These findings indicate that, with respect to PRAI-InGPS, similarity to E. coli among the organisms examined is in the following order: (E. aerogenes, S. typhimurium, P. vulgaris) greater than (S. marcescens, E. carotovora).     

Temperature sensitivity of the penicillin-induced autolysis mechanism in nongrowing cultures of Escherichia coli.

The effect of incubation temperature on the ampicillin-induced autolysis of nongrowing Escherichia coli was determined. The autolysis mechanisms in amino acid-deprived relA mutant cells treated with chloramphenicol were temperature sensitive. This temperature-sensitive autolysis was demonstrated in three independent ways: turbidimetric determinations, viable cell counts, and solubilization of radiolabeled peptidoglycan.     

Catalytic domains of carbamyl phosphate synthetase. Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase

We present evidence that cysteine 269 of the small subunit of Escherichia coli carbamyl phosphate synthetase is essential for the hydrolysis of glutamine. When cysteine 269 is replaced with glycine or with serine by site-directed mutagenesis of the carA gene, the resulting enzymes are unable to catalyze carbamyl phosphate synthesis with glutamine as nitrogen donor. Even though the glycine 269, and particularly the serine 269 enzyme bind significant amounts of glutamine, neither glycine 269 nor serine 269 can hydrolyze glutamine. The mutations at cysteine 269 do not affect carbamyl phosphate synthesis with NH3 as substrate. The NH3-dependent activity of the mutant enzymes was equal to that of wild-type. Measurements of Km indicate that the enzyme uses unionized NH3 rather than ammonium ion as substrate. The apparent Km for NH3 of the wild-type enzyme is calculated to be about 5 mM, independent of pH. The substitution of cysteine 269 with glycine or with serine results in a decrease of the apparent Km value for NH3 from 5 mM with the wild-type to 3.9 mM with the glycine, and 2.9 mM with the serine enzyme. Neither the glycine nor the serine mutation at position 269 affects the ability of the enzyme to catalyze ATP synthesis from ADP and carbamyl phosphate. Allosteric properties of the large subunit are also unaffected. However, substitution of cysteine 269 with glycine or with serine causes an 8- and 18-fold stimulation of HCO-3 -dependent ATPase activity, respectively. The increase in ATPase activity and the decrease in apparent Km for NH3 provide additional evidence for an interaction of the glutamine binding domain of the small subunit with one of the two known ATP sites of the large subunit.     

Inactivation of Escherichia coli glutamine synthetase by thiourea trioxide

Incubation of Escherichia coli glutamine synthetase with thiourea trioxide resulted in partial inactivation of the enzyme. This reagent specifically modifies lysine residues to form homoarginine. By amino acid analysis 2.3 ± 0.3 residues of homoarginine are produced per enzyme subunit after treatment with thiourea trioxide. Previously we determined that thiourea dioxide totally inactivated glutamine synthetase and modified both lysine and histidine residues (J. Colanduoni and J. J. Villafranca (1985) J. Biol. Chem. 260, 15,042–15,050). Thiourea trioxide reacted with the same lysine residues of glutamine synthetase as thiourea dioxide. The K_m values for the thiourea trioxide modified enzyme were determined and are 210 ± 30 μm and 10 ± 1 mm for ATP and glutamate, respectively. Both values are about threefold higher than for native enzyme assayed under the same conditions. Fluorescence titrations of native and thiourea trioxide labeled enzyme showed that ATP binding was virtually unchanged by the modification while glutamate and methionine sulfoximine bound about twofold more weakly to the modified enzyme.     

Induction of autolysis in nongrowing Escherichia coli

Unless relaxation of the stringent response is achieved, all nongrowing bacteria rapidly develop resistance to autolysis induced by a variety of agents, including all classes of cell wall synthesis inhibitors. We now describe inhibitors of cell wall synthesis which were unusual in that they could continue to effectively induce autolysis in relA+ Escherichia coli even after prolonged amino acid starvation. The process of cell wall degradation seems to be catalyzed by similar hydrolytic enzymes in nongrowing and growing cells, yet the activity of these new agents capable of inducing autolysis in the nongrowing relA+ cells did not involve relaxation of RNA or peptidoglycan synthesis. We propose that the suppression of autolysis characteristic of nongrowing cells can be bypassed by a novel mechanism of autolytic triggering which is independent of the relA locus.     

Amino-terminal sequences of indoleglycerol phosphate synthetase of Escherichia coli and Salmonella typhimurium.

The partial sequences of the first 40 residues of indoleglycerol phosphate synthetase of Escherichia coli and Salmonella typhimurium were determined, and three amino acid differences were observed among the 38 residues compared.     

The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate, which is subsequently employed in both the pyrimidine and arginine biosynthetic pathways. The reaction mechanism is known to proceed through at least three highly reactive intermediates: ammonia, carboxyphosphate, and carbamate. In keeping with the fact that the product of CPS is utilized in two competing metabolic pathways, the enzyme is highly regulated by a variety of effector molecules including potassium and ornithine, which function as activators, and UMP, which acts as an inhibitor. IMP is also known to bind to CPS but the actual effect of this ligand on the activity of the enzyme is dependent upon both temperature and assay conditions. Here we describe the three-dimensional architecture of CPS with bound IMP determined and refined to 2.1 A resolution. The nucleotide is situated at the C-terminal portion of a five-stranded parallel beta-sheet in the allosteric domain formed by Ser(937) to Lys(1073). Those amino acid side chains responsible for anchoring the nucleotide to the polypeptide chain include Lys(954), Thr(974), Thr(977), Lys(993), Asn(1015), and Thr(1017). A series of hydrogen bonds connect the IMP-binding pocket to the active site of the large subunit known to function in the phosphorylation of the unstable intermediate, carbamate. This structural analysis reveals, for the first time, the detailed manner in which CPS accommodates nucleotide monophosphate effector molecules within the allosteric domain.     

Growth of Escherichia coli in phosphate limited cultures : Measurements and modelling

The batch cultivation ofEscherichia coli limited by inorganic phosphate shows an exponential growth phase followed by a linear phase when the phosphate in the medium is exhausted. Data analysis and modelling of batch cultivation with different initial concentrations of the limiting factor give information about an exchangeable phosphate store, with RNA as the main component, and a non-exchangeable store.     

Understanding and improving NADPH-dependent reactions by nongrowing Escherichia coli cells

We have shown that whole Escherichia coli cells overexpressing NADPH-dependent cyclohexanone monooxygenase carry out a model Baeyer-Villiger oxidation with high volumetric productivity (0.79 g epsilon-caprolactone/L.h ) under nongrowing conditions (Walton, A. Z.; Stewart, J. D. Biotechnol. Prog. 2002, 18, 262-268). This is approximately 20-fold higher than the space-time yield for reactions that used growing cells of the same strain. Here, we show that the intracellular stability of cyclohexanone monooxygenase and the rate of substrate transport across the cell membrane were the key limitations on the overall reaction duration and rate, respectively. Directly measuring the levels of intracellular nicotinamide cofactors under bioprocess conditions suggested that E. coli cells could support even more efficient NADPH-dependent bioconversions if a more suitable enzyme-substrate pair were identified. This was demonstrated by reducing ethyl acetoacetate with whole cells of an E. coli strain that overexpressed an NADPH-dependent, short-chain dehydrogenase from baker's yeast (Saccharomyces cerevisiae). Under glucose-fed, nongrowing conditions, this reduction proceeded with a space-time yield of 2.0 g/L.h and a final product titer of 15.8 g/L using a biocatalyst:substrate ratio (g/g) of only 0.37. These values are significantly higher than those obtained previously. Moreover, the stoichiometry linking ketone reduction and glucose consumption (2.3 +/- 0.1) suggested that the citric acid cycle supplied the bulk of the intracellular NADPH under our process conditions. This information can be used to improve the efficiency of glucose utilization even further by metabolic engineering strategies that increase carbon flux through the pentose phosphate pathway.     

Biochemical and genetic characterization of a carbamyl phosphate synthetase mutant of Escherichia coli K12.

An unusual Escherichia coli K12 mutant for carbamyl phosphate synthetase is described. The mutation was generated by bacteriophage MUI insertion and left a 5% residual activity of the enzyme using either ammonia or glutamine as donors. The mutation is recessive to the wild-type allele and maps at or near the pyrA gene, but the mutant requires only arginine and not uracil for growth. By a second block in the pyrB gene it was possible to shift the accumulated carbamyl phosphate to arginine biosynthesis. The Km values and the levels of ornithine activation and inhibition by UMP were normal in the mutant enzyme.     

Allosteric control of the oligomerization of carbamoyl phosphate synthetase from Escherichia coli

Carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by the metabolites ornithine, IMP, and UMP. Ornithine and IMP function as activators, whereas UMP is an inhibitor. CPS undergoes changes in the state of oligomerization that are dependent on the protein concentration and the binding of allosteric effectors. Ornithine and IMP promote the formation of an (alphabeta)4 tetramer while UMP favors the formation of an (alphabeta)2 dimer. The three-dimensional structure of the (alphabeta)4 tetramer has unveiled two regions of molecular contact between symmetry-related monomeric units. Identical residues within two pairs of allosteric domains interact with one another as do twin pairs of oligomerization domains. There are thus two possible structures for an (alphabeta)2 dimer: an elongated dimer formed at the interface of two allosteric domains and a more compact dimer formed at the interface between two oligomerization domains. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (alphabeta)4 tetramer through disruption of the molecular binding interactions between monomeric units. When Leu-421 (located in the oligomerization domain) was mutated to a glutamate residue, CPS formed an (alphabeta)2 dimer in the presence of ornithine, UMP, or IMP. In contrast, when Asn-987 (located in the allosteric binding domain) was mutated to an aspartate, an (alphabeta) monomer was formed regardless of the presence of any allosteric effectors. These results are consistent with a model for the structure of the (alphabeta)2 dimer that is formed through molecular contact between two pairs of allosteric domains. Apparently, the second interaction, between pairs of oligomerization domains, does not form until after the interaction between pairs of allosteric domains is formed. The binding of UMP to the allosteric domain inhibits the dimerization of the (alphabeta)2 dimer, whereas the binding of either IMP or ornithine to this same domain promotes the dimerization of the (alphabeta)2 dimer. In the oligomerization process, ornithine and IMP must exert a conformational alteration on the oligomerization domain, which is approximately 45 A away from their site of binding within the allosteric domain. No significant dependence of the specific catalytic activity on the protein concentration could be detected, and thus the effects induced by the allosteric ligands on the catalytic activity and the state of oligomerization are unlinked from one another.     

An aminoacyl-tRNA synthetase complex in Escherichia coli.

Aminoacyl-tRNA synthetases from several strains of Escherichia coli are shown to elute as a high-molecular-weight complex on 6% agarose columns (Bio-Gel A-5M). In contrast, very little synthetase activity was observed in such complexes on Sephadex G-200 columns, suggesting that these enzymes may interact with or are dissociated during chromatography on dextran. The size of the complex observed on Bio-Gel A-5M was influenced by the method of cell breakage and the salt concentrations present in buffers. The largest complexes (greater than 1,000,000 daltons) were seen with cells broken with a freeze press, whereas with sonicated preparations the average size of the complex was about 400,000 daltons. Extraction of synthetases at 0.15 M NaCl, to mimic physiological salt concentrations, also resulted in high-molecular-weight complexes, as demonstrated by both agarose gel filtration and ultracentrifugation analysis. Evidence is presented that dissociation of some synthetases does occur in the presence of higher salt levels (0.4 M NaCl). Partial purification of the synthetase complex on DEAE-Sephacel was accomplished with only minor dissociation of individual synthetases. These data suggest that a complex(es) of aminoacyl-tRNA synthetase does exist in bacterial cells, just as in eucaryotes, and that the complex may have escaped earlier detection due to its fragility during isolation.     

Monomeric structure of glutamyl-tRNA synthetase in Escherichia coli.

An investigation of the subunit structure of glutamyl-tRNA synthetase (EC 6.1.1.17) from Escherichia coli indicates that this enzyme is a monomer. The enzyme purified to apparent homogeneity is a single polypeptide chain with a molecular weight of 62,000 ± 3,000 and K^Glu_m ? 50 μM in the aminoacylation reaction. Analytical gel electrophoretic procedures were used to determine the molecular weight of species exhibiting glutamyl-tRNA synthetase activity in freshly prepared extracts of several strains of E. coli, which had been grown under various nutritional conditions and harvested at different stages of growth. In all cases, glutamyl-tRNA synthetase activity was associated with a protein having about the same molecular weight and K^Glu_m as the purified enzyme. Thus, no evidence of an oligomeric form of glutamyl-tRNA synthetase with a greater affinity for l-glutamate was obtained, in contrast to a previous report of J. Lapointe and D. Söll (J. Biol. Chem.247, 4966–4974, 1972).     

Crystals of glutamine synthetase from Escherichia coli.

Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are $\text{a = 140 ± 2}\text{A}\text{̊}$, and $\text{c = 148 ± 2}\text{A}\text{̊}$. The Laue symmetry group is $\text{3}\text{̄}$m P3₁.     

EntG activity of Escherichia coli enterobactin synthetase.

The last steps in the biosynthesis of the Escherichia coli siderophore enterobactin (Ent) are carried out by Ent synthetase, a multienzyme complex believed to be composed of the entD, -E, -F, and -G products (EntD to -G). However, sequencing data showed that there is no separate entG gene and, unlike EntD to -F, no distinct EntG polypeptide has been identified. In this study, genetic, biochemical, and immunological approaches were used to study the anomalies associated with EntG activity. Two plasmids, pJS43 and pJS100, were isolated that had mutations resulting in truncated EntB proteins; both had the phenotype EntB+ EntG-. PJS43 had a Tn5 inserted 198 bp from the entB termination codon, and pJS100 had the last 25 codons of entB deleted. Plasmids isolated with Tn5 insertions in the 5' half of entB had the phenotype EntB- EntG+. These latter Tn5 mutations were EntB- EntG- when moved to the bacterial chromosome. Polyclonal antiserum was prepared and shown to react only with intact EntB in Western immunoblots. Addition of anti-EntB antiserum to Ent synthetase assays resulted in complete inhibition of enzyme activity, whereas preimmune serum had no effect. Lastly, AN462, the type strain for entG which was derived by Mu insertion and which has the phenotype EntB-G-A-, was characterized. Southern blot data showed a Mu insertion, presumably with polar effects, in the vicinity of the 5' end of entB. In summary, EntG activity was found to be encoded by the entB 3' terminus. The evidence, while not rigorously eliminating the possibility that a separate EntG polypeptide exists, strongly supports the idea that EntB is a bifunctional protein.