Homoserine kinase from Escherichia coli K12.

Homoserine kinase was purified to apparent homogeneity from a derepressed strain of Escherichia coli K12, using standard fractionation techniques. It is a dimer (Mr = 60000) composed of apparently identical polypeptide chains (Mr = 29000). Its amino acid composition and N-terminal sequence have been determined. L-Threonine is a competitive inhibitor of the substrate L-homoserine; this inhibition is straighforward and shows no sign of co-operativity. Evidence is presented that homoserine and threonine bind to the same site of this non-allosteric enzyme. The binding of homoserine and threonine can also be studied by difference spectroscopy; the latter studies reveal an unexpected effect of magnesium ions, which might be the basis for the unusual high Mg2+ requirement for optimal enzyme reaction.     

Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12.

Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity. The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression. The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants. Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR. By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000. The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E. coli chromosome between the structural genes proC and purE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated. The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme.     

L-threonine dehydrogenase from Escherichia coli K-12: thiol-dependent activation by Mn2+

Addition of 1 mM Mn2+ to all solutions in the final chromatographic step used to purify L-threonine dehydrogenase (L-threonine:NAD+ oxidoreductase, EC 1.1.1.103) from extracts of Escherichia coli K-12 routinely provides 30-40 mg of pure enzyme per 100 g wet weight of cells with specific activity = 20-30 units/mg. Enzyme dialyzed exhaustively against buffers containing Chelex-100 resin has a specific activity = 8 units/mg and contains 0.003 or 0.02 mol of Mn2+/mol of enzyme as determined by radiolabeling studies with 54Mn2+ or by atomic absorption spectroscopy, respectively. Dehydrogenase activity is completely abolished by low concentrations of either Hg2+ or Ag+; of a large spectrum of other metal ions tested, only Mn2+ and Cd2+ have an activating effect. Activation of threonine dehydrogenase by Mn2+ is thiol-dependent and is saturable with an activation Kd = 9.0 microM and a Vmax = 105 units/mg. Stoichiometry of Mn2+ binding was found to be 0.86 mol of Mn2+/mol of enzyme subunit with a dissociation constant (Kd) = 8.5 microM. Mn2+ appears to interact directly with threonine dehydrogenase; gel filtration studies with the dehydrogenase plus 54Mn2+ in the presence of either NAD+, NADH, L-threonine, or combinations thereof show that only Mn2+ coelutes with the enzyme whereas all other ligands elute in the salt front and the stoichiometry of the dehydrogenase-Mn2+ interaction is not affected in any instance. A theoretical curve fit to data for the pH-activity profile of Mn2+-saturated enzyme has a pKa = 7.95 for one proton ionization. The data establish L-threonine dehydrogenase of E. coli to be a metal ion activated enzyme.     

Homoserine kinase of Escherichia coli: kinetic mechanism and inhibition by L-aspartate semialdehyde

The kinetic mechanism of homoserine kinase, purified to homogeneity from Escherichia coli, was examined by initial velocity techniques at pH 7.6. Whereas ATP displayed normal Michaelis-Menten saturation kinetics (Km = 0.2 mM), L-homoserine showed hyperbolic saturation kinetics only up to a concentration of 0.75 mM (Km = 0.15 mM). Above this concentration, L-homoserine caused marked but partial inhibition (Ki approximately 2 mM). The kinetic data indicated that the addition of substrates to homoserine kinase occurs by a preferred order random mechanism, with ATP preferentially binding before L-homoserine. When the ATP concentration was varied at several fixed inhibitory concentrations of L-homoserine, the resulting inhibition pattern indicated hyperbolic mixed inhibition. This suggested a second binding site for L-homoserine. L-Aspartate semialdehyde, an amino acid analog of L-homoserine, proved to be an alternative substrate of homoserine kinase (Km = 0.68 mM), and was subsequently used as a probe of its kinetic mechanism. In aqueous solution, at pH 7.5, this analog was found to exist predominantly (ca 85%) as its hydrated species. When examined as an inhibitor of the physiological reaction, L-aspartate semialdehyde showed mixed inhibition versus both L-homoserine and ATP. Although the pH profiles for the binding of L-homoserine as a substrate (Km) and as an inhibitor (Ki) were identical, the kinetic data were best fit to a two-site model, with separate catalytic and inhibitory sites for L-homoserine.     

Formation of D-1-amino-2-propanol from L-threonine by enzymes from Escherichia coli K-12

Escherichia coli K-12 cells contain two dehydrogenases which in sequence catalyze the net conversion of L-threonine to the D-isomer of 1-amino-2-propanol. These two enzymes are L-threonine dehydrogenase (L-threonine + NAD⁺ → aminoacetone + CO₂ + NADH + H⁺) and D-1-amino-2-propanol dehydrogenase (aminoacetone + NADH + H⁺D-1-amino-2-propanol + NAD⁺). Each enzyme has been obtained in purified form free of the other; the nature of the reaction catalyzed by the latter dehydrogenase alone and in a coupled system with the former enzyme has been studied. The results provide an explanation on the enzymological level for the utilization of L-threonine by cell suspensions of certain microorganisms for the biosynthesis of the D-1-amino-2-propanol moiety of Vitamin B₁₂.     

gamma-Glutamyltranspeptidase from Escherichia coli K-12: purification and properties.

gamma-Glutamyltranspeptidase (GGT) (EC 2.3.2.2) was purified from the periplasmic fraction of Escherichia coli K-12 to electrophoretic homogeneity. The final purification step, chromatofocusing, gave two protein peaks showing GGT activity (fractions A and B). The major heavy fraction (fraction A) consisted of two different subunits, with molecular weights of 39,200 and 22,000. The minor light fraction (fraction B) consisted of those with molecular weights of 38,600 and 22,000. Fraction A catalyzes the hydrolysis and transpeptidation of all gamma-glutamyl compounds tested, but it prefers basic amino acids and aromatic amino acids as acceptors. The apparent Km values for glutathione and gamma-glutamyl-p-nitroanilide as gamma-glutamyl donors in the transpeptidation reaction were both 35 microM, and those for glycylglycine and L-arginine as acceptors were 0.59 and 0.21 M, respectively. The enzyme was inhibited by some amino acids and by protease inhibitors and affinity-labeling reagents for GGT. The temperature stability of the purified GGT supports our hypothesis that E. coli GGT is synthesized only at lower temperature rather than that the synthesized GGT is degraded or inactivated at higher temperature.     

Independent regulation of transport and biosynthesis of arginine in Escherichia coli K-12.

From an arginine auxotrophic strain, a mutant was isolated which is able to utilize d-arginine as a source of l-arginine and shows a high sensitivity to inhibition of growth by canavanine. Transport studies revealed a four- to five-fold increased uptake of arginine and ornithine in cells from the mutant strain. The kinetics of entry of arginine and ornithine evidenced elevated maximal influx values for the arginine- and ornithine-specific transport systems. A close parallel between arginine transport activity and arginine binding activity with one arginine-specific binding periplasmic protein in the mutant strongly suggests that such binding protein is a component of the arginine-specific permease. The affinity between arginine and the binder, isolated from the mutant cells, as well as the electrophoretic mobility of the protein, remain unchanged. The enhanced transport activity of arginine and ornithine with mutant cells is insensitive to repression by arginine or ornithine, whereas the biosynthesis of arginine-forming enzymes is normally repressible. When transport activity was examined in strains with mutations leading to derepression of arginine biosynthesis, the regulation of arginine transport was found to be normal. These studies support the conclusion that arginine transport and arginine biosynthesis, in Escherichia coli K-12, are not regulated in a concerted manner, although both systems may have components in common.     

Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12.

The anaerobic regulation of the gene encoding pyruvate formate-lyase from Escherichia coli was investigated. Expression of a pfl'-'lacZ protein fusion demonstrated that the gene is subject to a 12-fold anaerobic induction which can be stimulated a further 2-fold by the addition of pyruvate to the growth medium. Construction of a strain deleted for pfl verified that either pyruvate or a metabolite of glycolysis functions as an inducer of pfl gene expression. Complete anaerobic induction required the presence of a functional fnr gene product. However, the dependence was not absolute since a two- to threefold anaerobic induction could still be observed in an fnr mutant. These results could be confirmed immunologically by analyzing the levels of pyruvate formate-lyase protein present in cells grown under various conditions. It was also shown that pfl'-'lacZ expression was partially repressed by nitrate and that this repression was mediated by the narL gene product.     

Studies of homogeneous "biosynthetic" L-threonine dehydratase from Escherichia coli K-12. Some kinetic properties and molecular multiplicity.

"Biosynthetic" L-threonine dehydratase (EC 4.2.1.16) was purified to a homogeneous state with 29% yield of total activity from Escherichia coli K-12. The homogeneity of the enzyme was shown by polyacrylamide gel disc electrophoresis in the presence of dodecyl sulphate. The enzyme consisted of equal subunits having a molecular weight of about 57 000. The polyacrylamide gel disc electrophoresis has shown that the native enzyme consisted of a set of oligomeric forms. The multiplicity of molecular organization of the enzyme was reflected in complicated kinetic behaviour: at pH greater than 9 on the plots of initial reaction rate (v) versus initial substrate concentration ([S]o) there were four inflexion points (two intermediate plateaux), the position and deepness of which depended on enzyme concentration. At pH 8.3 on the v versus [S]o plots appeared two inflexion points (one intermediate plateu), the position of which practically did not depend on enzyme concentration in the reaction mixture, but strongly depended on the enzyme concentration in the stock solution. Repeated polyacrylamide gel disc electrophoresis of several oligomeric forms, isolated by the first electrophoresis, has shown that the oligomeric forms underwent a slow polymerization. It was suggested that "biosynthetic" L-threonine dehydratase from E. coli K-12 is a set of multiple oligomeric forms, having different kinetic parameters. Probably, each form of the enzyme has a "simple" kinetics characterized by hyperbolic or sigmoidal shape of v versus [S]o plots. The rate of equilibrium installation between the oligomeric forms was small in comparison with the enzyme reaction velocity, that lead to the complex kinetic curves, appearing as a result of summing up of the kinetics inherent to theindividual forms.     

Mutant of Escherichia coli K-12 defective in D-glucosamine biosynthesis

A mutant was isolated from a derivative of Escherichia coli K-12 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. This mutant contained normal levels of 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10), but no detectable activity of l-glutamine:d-fructose-6-phosphate amino-transferase (EC 2.6.1.16). It required either N-acetyl-d-glucosamine or d-glucosamine for growth, and went into rapid lysis when the supply of these compounds was exhausted. In medium containing 11% sucrose, the cells were converted into spheroplasts in the absence of d-glucosamine.     

Transcriptional regulation of katE in Escherichia coli K-12

Escherichia coli produces two distinct species of catalase, hydroperoxidases I and II, which differ in kinetic properties and regulation. To further examine catalase regulation, a lacZ fusion was placed into one of the genes that is involved in catalase synthesis. Transductional mapping revealed the fusion to be either allelic with or very close to katE, a locus which together with katF controls the synthesis of the aerobically inducible hydroperoxidase (hydroperoxidase II). katE was expressed under anaerobic conditions at levels that were approximately one-fourth of those found in aerobically grown cells and was found to be induced to higher levels in early-stationary-phase cells relative to levels of exponentially growing cells under both anaerobic and aerobic conditions. katE was fully expressed in air and was not further induced when the growth medium was sparged with 100% oxygen. Expression of katE was unaffected by the addition of hydrogen peroxide or by the presence of additional lesions in oxyR or sodA, indicating that it is not part of the oxyR regulon. When katF::Tn10 was introduced into a katE::lacZ strain, beta-galactosidase synthesis was largely eliminated and was no longer inducible, suggesting that katF is a positive regulator of katE expression.     

Purification and properties of a kinase from Escherichia coli K-12 that phosphorylates two periplasmic transport proteins

The phosphorylation in vivo and in vitro of the arginine-ornithine and the lysine-arginine-ornithine (LAO) periplasmic transport proteins of Escherichia coli K-12 was previously reported (Celis, R. T. F. (1984) Eur. J. Biochem. 145, 403-411). The phosphorylative reaction required ATP (as a direct energy donor), Mg2+, and a kinase that can be released by osmotic shock treatment of the cells. The enzyme was purified to electrophoretic homogeneity. The enzyme exhibited an ATPase activity and a kinase activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave an apparent molecular weight of 43,000 for the enzyme. The native protein showed the same molecular weight, suggesting that the protein is a monomer. The protein showed an apparent isoelectric point of 4.8 on isoelectric focusing. The two enzymatic reactions required a divalent cation and the apparent Km value for Mg2+ for the kinase activity was 0.5 mM. Mn2+ and Co2+ served as well as Mg2+, whereas Zn2+ and Ca2+ did not support activity. The ATPase activity of the enzyme yielded an apparent Km value for ATP of 50 microM. A similar value, Km of 100 microM, was calculated for the kinase activity with different concentrations of ATP. The enzyme showed a pH optimum of 7.3.     

Purification and properties of inosine-guanosine phosphorylase from Escherichia coli K-12.

A xanthosine-inducible enzyme, inosine-guanosine phosphorylase, has been partially purified from a strain of Escherichia coli K-12 lacking the deo-encoded purine nucleoside phosphorylase. Inosine-guanosine phosphorylase had a particle weight of 180 kilodaltons and was rapidly inactivated by p-chloromercuriphenylsulfonic acid (p-CMB). The enzyme was not protected from inactivation by inosine (Ino), 2'-deoxyinosine (dIno), hypoxanthine (Hyp), Pi, or alpha-D-ribose-1-phosphate (Rib-1-P). Incubating the inactive enzyme with dithiothreitol restored the catalytic activity. Reaction with p-CMB did not affect the particle weight. Inosine-guanosine phosphorylase was more sensitive to thermal inactivation than purine nucleoside phosphorylase. The half-life determined at 45 degrees C between pH 5 and 8 was 5 to 9 min. Phosphate (20 mM) stabilized the enzyme to thermal inactivation, while Ino (1 mM), dIno (1 mM), xanthosine (Xao) (1 mM), Rib-1-P (2 mM), or Hyp (0.05 mM) had no effect. However, Hyp at 1 mM did stabilize the enzyme. In addition, the combination of Pi (20 mM) and Hyp (0.05 mM) stabilized this enzyme to a greater extent than did Pi alone. Apparent activation energies of 11.5 kcal/mol and 7.9 kcal/mol were determined in the phosphorolytic and synthetic direction, respectively. The pH dependence of Ino cleavage or synthesis did not vary between 6 and 8. The substrate specificity, listed in decreasing order of efficiency (V/Km), was: 2'-deoxyguanosine, dIno, guanosine, Xao, Ino, 5'-dIno, and 2',3'-dideoxyinosine. Inosine-guanosine phosphorylase differed from the deo operon-encoded purine nucleoside phosphorylase in that neither adenosine, 2'-deoxyadenosine, nor hypoxanthine arabinoside were substrates or potent inhibitors. Moreover, the E. coli inosine-guanosine phosphorylase was antigenically distinct from the purine nucleoside phosphorylase since it did not react with any of 14 monoclonal antisera or a polyvalent antiserum raised against deo-encoded purine nucleoside phosphorylase.     

Repression of aromatic amino acid biosynthesis in Escherichia coli K-12

Mutants of Escherichia coli K-12 were isolated in which the synthesis of the following, normally repressible enzymes of aromatic biosynthesis was constitutive: 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetases (phe and tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A. In the wild type, DAHP synthetase (phe) was multivalently repressed by phenylalanine plus tryptophan, whereas DAHP synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A were repressed by tyrosine. DAHP synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase were also repressed by phenylalanine in high concentration (10(-3)m). Besides the constitutive synthesis of DAHP synthetase (phe), the mutants had the same phenotype as strains mutated in the tyrosine regulatory gene tyrR. The mutations causing this phenotype were cotransducible with trpA, trpE, cysB, and pyrF and mapped in the same region as tyrR at approximately 26 min on the chromosome. It is concluded that these mutations may be alleles of the tyrR gene and that synthesis of the enzymes listed above is controlled by this gene. Chorismate mutase P and prephenate dehydratase activities which are carried on a single protein were repressed by phenylalanine alone and were not controlled by tyrR. Formation of this protein is presumed to be controlled by a separate, unknown regulator gene. The heat-stable phenylalanine transaminase and two enzymes of the common aromatic pathway, 5-dehydroquinate synthetase and 5-dehydroquinase, were not repressible under the conditions studied and were not affected by tyrR. DAHP synthetase (trp) and tryptophan synthetase were repressed by tryptophan and have previously been shown to be under the control of the trpR regulatory gene. These enzymes also were unaffected by tyrR.     

Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12.

[3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli.     

Regulation of tyrosine and phenylalanine biosynthesis in Escherichia coli K-12: properties of the tyrR gene product

A spontaneous amber tyrR mutant has been isolated in which constitutive synthesis of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (tyr) and DAHP synthetase (phe) is suppressible by supC(-), supD(-), supF(-) and supU(-). This finding suggests the tyrR gene product is a protein. Derepression of DAHP synthetase (phe) in this and in seven other spontaneous tyrR mutants and in four Mu-1-induced tyrR mutants provides further evidence for the involvement of the tyrR gene product in phenylalanine biosynthesis. Evidence that the tyrR product is a component of repressor, rather than an enzyme involved in its synthesis or modification, comes from a study of a temperature-sensitive tyrR mutant. This mutant is of the thermolabile type, since derepression occurs rapidly and in the presence and absence of growth.