Adenosine diphosphate glucose pyrophosphatase: A plastidial phosphodiesterase that prevents starch biosynthesis

A distinct phosphodiesterasic activity (EC 3.1.4) was found in both mono- and dicotyledonous plants that catalyzes the hydrolytic breakdown of ADPglucose (ADPG) to produce equimolar amounts of glucose-1-phosphate and AMP. The enzyme responsible for this activity, referred to as ADPG pyrophosphatase (AGPPase), was purified over 1,100-fold from barley leaves and subjected to biochemical characterization. The calculated K(eq)' (modified equilibrium constant) value for the ADPG hydrolytic reaction at pH 7.0 and 25 degrees C is 110, and its standard-state free-energy change value (DeltaG') is -2.9 kcal/mol (1 kcal = 4.18 kJ). Kinetic analyses showed that, although AGPPase can hydrolyze several low-molecular weight phosphodiester bond-containing compounds, ADPG proved to be the best substrate (K(m) = 0.5 mM). P(i) and phosphorylated compounds such as 3-phosphoglycerate, PP(i), ATP, ADP, NADP(+), and AMP are inhibitors of AGPPase. Subcellular localization studies revealed that AGPPase is localized exclusively in the plastidial compartment of cultured cells of sycamore (Acer pseudoplatanus L.), whereas it occurs both inside and outside the plastid in barley endosperm. In this paper, evidence is presented that shows that AGPPase, whose activity declines concomitantly with the accumulation of starch during development of sink organs, competes with starch synthase (ADPG:1,4-alpha-d-glucan 4-alpha-d-glucosyltransferase; EC) for ADPG, thus markedly blocking the starch biosynthesis.     

Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli

Bacterial aromatic polyketides are important therapeutic compounds including front line antibiotics and anticancer drugs. It is one of the last remaining major classes of natural products of which the biosynthesis has not been reconstituted in the genetically superior host Escherichia coli. Here, we demonstrate the engineered biosynthesis of bacterial aromatic polyketides in E. coli by using a dissected and reassembled fungal polyketide synthase (PKS). The minimal PKS of the megasynthase PKS4 from Gibberella fujikuroi was extracted by using two approaches. The first approach yielded a stand-alone Ketosynthase (KS)_malonyl-CoA:ACP transferase (MAT) didomain and an acyl-carrier protein (ACP) domain, whereas the second approach yielded a compact PKS (PKS_WJ) that consists of KS, MAT, and ACP on a single polypeptide. Both minimal PKSs produced nonfungal polyketides cyclized via different regioselectivity, whereas the fungal-specific C2-C7 cyclization mode was not observed. The kinetic properties of the two minimal PKSs were characterized to confirm both PKSs can synthesize polyketides with similar efficiency as the parent PKS4 megasynthase. Both minimal PKSs interacted effectively with exogenous polyketide cyclases as demonstrated by the synthesis of predominantly PK8 3 or NonaSEK4 6 in the presence of a C9-C14 or a C7-C12 cyclase, respectively. When PKS_WJ and downstream tailoring enzymes were expressed in E. coli, the expected nonaketide anthraquinone SEK26 was recovered in good titer. High-cell density fermentation was performed to demonstrate the scale-up potential of the in vivo platform for the biosynthesis of bacterial polyketides. Using engineered fungal PKSs can therefore be a general approach toward the heterologous biosynthesis of bacterial aromatic polyketides in E. coli.     

Primary structure of the Escherichia coli ribonucleoside diphosphate reductase operon.

The nucleotide sequence of the Escherichia coli K-12 DNA comprising the operon for the structural genes of the subunits of ribonucleotide diphosphate reductase has been determined. The DNA sequenced maps at 48.5 minutes on the E. coli chromosome and includes a total length of 8557 nucleotides. An open reading frame between nucleotides 3506 and 5834, encoding a 776-amino acid polypeptide chain with a molecular weight of 87,532, has been identified as the nrdA gene. An open reading frame between nucleotides 6012 and 7139, encoding a 375-amino acid polypeptide with a molecular weight of 43,466, has been identified as the nrdB gene. The sequences reveal not only the primary structures for both subunits, but also some interesting aspects of potential regulatory sites.     

Escherichia coli nucleoside diphosphate kinase is a uracil-processing DNA repair nuclease

Escherichia coli nucleoside diphosphate kinase (eNDK) is an XTP:XDP phosphotransferase that plays an important role in the regulation of cellular nucleoside triphosphate concentrations. It is also one of several recently discovered DNases belonging to the NM23/NDK family. E. coli cells disrupted in the ndk gene display a spontaneous mutator phenotype, which has been attributed to the mutagenic effects of imbalanced nucleotide pools and errors made by replicative DNA polymerases. Another explanation for the increased mutation rates is that endk- cells lack the nuclease activity of the NDK protein that is essential for a DNA repair pathway. Here, we show that purified, cloned endk is a DNA repair nuclease whose substrate is uracil misincorporated into DNA. We have identified three new catalytic activities in eNDK that act sequentially to repair the uracil lesion: (i) uracil-DNA glycosylase that excises uracil from single-stranded and from U/A and U/G mispairs in double-stranded DNA; (ii) apyrimidinic endonuclease that cleaves double-stranded DNA as a lyase by forming a covalent enzyme-DNA intermediate complex with the apyrimidinic site created by the glycosylase; and (iii) DNA repair phosphodiesterase that removes 3'-blocking residues from the ends of duplex DNA. All three of these activities, as well as the nucleoside-diphosphate kinase, reside in the same protein. Based on these findings, we propose an editing function for eNDK as a mechanism by which the enzyme prevents mutations in DNA.     

Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli

Ansamycins such as rifamycin, ansamitocin, and geldanamycin are an important class of polyketide natural products. Their biosynthetic pathways are especially complex because they involve the formation of 3-amino-5-hydroxybenzoic acid (AHBA) followed by backbone assembly by a hybrid nonribosomal peptide synthetase/polyketide synthase. We have reconstituted the ability to synthesize 2,6-dimethyl-3,5,7-trihydroxy-7-(3'-amino-5'-hydroxyphenyl)-2,4-heptadienoic acid (P8/1-OG), an intermediate in rifamycin biosynthesis, in an extensively manipulated strain of Escherichia coli. The parent strain, BAP1, contains the sfp phosphopantetheinyl transferase gene from Bacillus subtilis, which posttranslationally modifies polyketide synthase and nonribosomal peptide synthetase modules. AHBA biosynthesis in this host required introduction of seven genes from Amycolatopsis mediterranei, which produces rifamycin, and Actinosynnema pretiosum, which produces ansamitocin. Because the four-module RifA protein (530 kDa) from the rifamycin synthetase could not be efficiently produced in an intact form in E. coli, it was genetically split into two bimodular proteins separated by matched linker pairs to facilitate efficient inter-polypeptide transfer of a biosynthetic intermediate. A derivative of BAP1 was engineered that harbors the AHBA biosynthetic operon, the bicistronic RifA construct and the pccB and accA1 genes from Streptomyces coelicolor, which enable methylmalonyl-CoA biosynthesis. Fermentation of this strain of E. coli yielded P8/1-OG, an N-acetyl P8/1-OG analog, and AHBA. In addition to providing a fundamentally new route to shikimate and ansamycin-type compounds, this result enables further genetic manipulation of AHBA-derived polyketide natural products with unprecedented power.     

Regulation of polyamine biosynthesis in Escherichia coli by basic proteins.

In Escherichia coli, the biosynthetic ornithine and arginine decarboxylases (EC 4.1.1.17 and 4.1.1.19, respectively) are responsible for the biosynthesis of polyamines from ornithine and arginine, respectively. When E. coli cells are grown in the presence of increasing amounts of polyamines, a progressive increase in the amount of antizyme 1 and antizyme 2 occurs. The amino acid compositions of antizymes 1 and 2 show them to be basic proteins; antizyme 1 has an amino acid composition similar to that of the E. coli histone-like protein HU and of the eukaryotic histone H2B; antizyme 2 is characterized by an unusually high arginine content. We find these proteins to be specific inhibitors of both the biosynthetic ornithine decarboxylase and the biosynthetic arginine decarboxylase. They do not inhibit the corresponding biodegradative ornithine and arginine decarboxylases, nor do they inhibit lysine decarboxylase or S-adenosylmethionine decarboxylase. These properties of the antizymes favor their function in the regulation of polyamine biosynthesis in E. coli. The ability of the purified antizymes to inhibit the ornithine and arginine decarboxylases is stabilized in acidic buffers and is lost upon prolonged exposure to solutions at neutral or basic pH.     

Osmotic regulation and the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

The membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic constituents containing 8-10 glucose units in a highly branched structure, linked by beta 1-2 and beta 1-6 bonds [Schneider, J. E. Reinhold, V., Rumley, M. K. & Kennedy, E. P. (1979) J. Biol. Chem. 254, 10135-10138]. The MDO are multiply substituted with sn-1-phosphoglycerol residues (derived from membrane phosphatidylglycerol) and with O-succinyl ester residues and, thus, are high anionic. Experiments in this paper offer evidence that the biosynthesis of MDO is an important aspects of osmoregulation in E. coli. Cells grown in medium of low osmolarity (ca. 50 mosM) synthesize 16 times more MDO than those grown in the same medium with 0.4 M NaCl. In cells grown in medium of low osmolarity, it appears that MDO is the principal source of fixed anion in the periplasmic space and, thus, acts to maintain the high osmotic pressure and Donnan membrane potential of the periplasmic compartment Regulation of MDO synthesis in response to changes in osmolarity of the medium appears to occur at the genetic level because the synthesis of new protein is needed to permit the production of MDO at high rates after shift of cells to medium of low osmolarity.     

Escherichia coli nucleoside diphosphate kinase does not act as a uracil-processing DNA repair nuclease

Escherichia coli nucleoside diphosphate kinase (Ndk) catalyzes ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the cognate diphosphate precursor. Recently, the Ndk polypeptide was reported to be a multifunctional base excision repair nuclease that processed uracil residues in DNA by acting sequentially as a uracil-DNA glycosylase inhibitor protein (Ugi)-sensitive uracil-DNA glycosylase, an apurinic/apyrimidiniclyase, and a 3'-phosphodiesterase [Postel, E. H. & Abramczyk, B. M. (2003) Proc. Natl. Acad. Sci. USA 100, 13247-13252]. Here we demonstrate that the E. coli Ndk polypeptide lacked detectable uracil-DNA glycosylase activity and, hence, was incapable of acting as a uracil-processing DNA repair nuclease. This finding was based on the following observations: (i) uracil-DNA glycosylase activity did not copurify with Ndk activity; (ii) Ndk purified from E. coli ung(-) cells showed no detectable uracil-DNA glycosylase activity; and (iii) Ndk failed to bind to a Ugi-Sepharose affinity column that tightly bound E. coli uracil-DNA glycosylase (Ung). Collectively, these observations demonstrate that the E. coli Ndk polypeptide does not possess inherent uracil-DNA glycosylase activity.     

Nonpathogenic Escherichia coli strain Nissle1917 prevents murine acute and chronic colitis

BACKGROUND: Nonpathogenic Escherichia coli strain Nissle1917 has been used as a probiotics in human inflammatory bowel disease; however, there are few reports examining its therapeutic effect on animal colitis models, and its therapeutic mechanisms remain unknown. The aim of this study was to elucidate the therapeutic effect and mechanism of Nissle1917 using murine acute and chronic colitis models. METHODS: Two models were used. (1) Acute model: colitis was induced by administration of 1.3% dextran sodium sulfate for 7 days. Nissle1917 or phosphate-buffered saline were orally administered for 10 days. Mice were killed at day 10, and the colonic lesions were assessed macro- and microscopically. (2) Chronic model: IL-10 mice were treated with Nissle1917 or phosphate-buffered saline for 8 weeks. After 8 weeks of treatment, mice were killed to assess the colonic lesions macro- and microscopically. In the acute dextran sodium sulfate colitis model, viable, heat-killed, or genomic DNA of Nissle1917 was orally administered for 10 days, and the therapeutic effect was assessed. RESULTS: In the acute model, Nissle1917 ameliorated body weight loss, disease activity index, and macro- and microscopic damage. In the chronic model, it also suppressed the mucosal inflammatory findings and histologic damages. Moreover, heat-killed Nissle1917 or its genomic DNA alone also ameliorated the acute DSS colitis and viable bacteria macro- and microscopically. CONCLUSIONS: Nonpathogenic E. coli strain Nissle1917 prevents both acute and chronic colitis, and its anti-inflammatory effect is exhibited not only by viable bacteria but also by heat-killed bacteria or its DNA.     

Pathway for lipid A biosynthesis in Arabidopsis thaliana resembling that of Escherichia coli

The lipid A moiety of Escherichia coli lipopolysaccharide is a hexa-acylated disaccharide of glucosamine that makes up the outer monolayer of the outer membrane. Arabidopsis thaliana contains nuclear genes encoding orthologs of key enzymes of bacterial lipid A biosynthesis, including LpxA, LpxC, LpxD, LpxB, LpxK and KdtA. Although structurally related lipid A molecules are found in most other gram-negative bacteria, lipid A and its precursors have not been directly detected in plants previously. However, homozygous insertional knockout mutations or RNAi knock-down constructs of Arabidopsis lpx and kdtA mutants revealed accumulation (or disappearance) of the expected monosaccharide or disaccharide lipid A precursors by mass spectrometry of total lipids extracted from 10-day old seedlings of these mutants. In addition, fluorescence microscopy of lpx-gfp fusions in transgenic Arabidopsis plants suggests that the Lpx and KdtA proteins are expressed and targeted to mitochondria. Although the structure of the lipid A end product generated by plants is still unknown, our work demonstrates that plants synthesize lipid A precursors using the same enzymatic pathway present in E. coli.     

Adenosine Triphosphate: Glutamine Synthetase Adenylyltransferase of Escherichia coli: Two Active Molecular Forms

Two active forms of purified ATP:glutamine synthetase adenylyl-transferase from Escherichia coli are apparent on polyacrylamide gel electrophoresis at pH 8. The slower migrating component, which is identical to the P(I)-protein fraction of the glutamine synthetase deadenylylating enzyme system, has S(20.w) congruent with 5.1 S and a molecular weight of about 130,000. The more rapidly migrating adenylyltransferase component has S(20.w) congruent with 4.0 S and a molecular weight of about 70,000. During storage at 4 degrees C, the larger adenylyltransferase component (P(I)) converts to the smaller active unit with a concomitant loss of both P(I) deadenylylating activity and soluble protein. It is concluded that the low-molecular weight form of the adenylyltransferase is a subunit of the deadenylylating P(I)-protein.     

Polyphosphate kinase as a nucleoside diphosphate kinase in Escherichia coli and Pseudomonas aeruginosa

Generation of a wide variety of nucleoside (and deoxynucleoside) triphosphates (NTPs) from their cognate nucleoside diphosphates (NDPs) is of critical importance in virtually every aspect of cellular life. Their function is fulfilled largely by the ubiquitous and potent nucleoside diphosphate kinase (NDK), most commonly using ATP as the donor. Considerable interest is attached to the consequence to a cell in which the NDK activity becomes deficient or overabundant. We have discovered an additional and possibly auxiliary NDK-like activity in the capacity of polyphosphate kinase (PPK) to use inorganic polyphosphate as the donor in place of ATP, thereby converting GDP and other NDPs to NTPs. This reaction was observed with the PPK activity present in crude membrane fractions from Escherichia coli and Pseudomonas aeruginosa as well as with the purified PPK from E. coli; the activity was absent from the membrane fractions obtained from E. coli mutants lacking the ppk gene. The order of substrate specificity for PPK was: ADP > GDP > UDP, CDP; activity with ADP was 2–60 times greater than with GDP, depending on the reaction condition. Although the transfer of a phosphate from polyphosphate to GDP by PPK to produce GTP was the predominant reaction, the enzyme also transferred a pyrophosphate group to GDP to form the linear guanosine 5′ tetraphosphate.     

Glucose and the Metabolism of Adenosine 3′:5′-Cyclic Monophosphate in Escherichia coli

Measurements of adenosine 3':5'-cyclic monophosphate (cAMP) concentrations have been made in Escherichia coli under various conditions. Different strains of E. coli accumulate different extracellular concentrations of cAMP (0.2-4 mum) at stationary phase. Mutation at the RNA control locus does not affect the accumulation pattern. Growth of the bacteria in minimalsalts medium leads to a greater accumulation of cAMP than growth in nutrient broth. Partition studies show that essentially all of the cAMP that is accumulated is found in the medium rather than in the cells. Kinetic studies show that most of the cAMP is formed coincidentally with exhaustion of glucose from the medium. Growth on high concentrations of glucose leads to inhibition of cAMP formation. Other carbon sources cannot substitute for glucose in this inhibitory effect. Measurements of enzyme activities indicate that glucose suppression of cAMP formation cannot be accounted for by a decreased activity of adenylate cyclase or an increased activity of cAMP phosphodiesterase (EC 3.1.3.7).     

Adenosine 3'':5''-cyclic monophosphate as mediator of catabolite repression in Escherichia coli.

Measurements of intracellular adenosine 3':5'-cyclic monophosphate (cAMP) concentrations in E. coli under a variety of conditions show that levels of this nucleotide are well correlated with the rate of synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in both catabolite repression and transient repression. These results, combined with extensive genetic and in vitro studies from a number of laboratories on the role of cAMP in E. coli, provide strong support for the concept that intracellular cAMP levels mediate the effects of catabolite and transient repression on rates on enzyme synthesis. Under all conditions studied, excretion can be described by a single rate constant, 2.1 min-1 at 37 degrees, indicating that intracellular levels cannot be regulated by alterations in the rate of cAMP excretion. Our data are fully consistent with the idea that carbon sources control intracellular cAMP levels by effects on its synthesis.     

Leukotriene antagonist FPL 57231 prevents the acute pulmonary effects of Escherichia coli endotoxin in cats

We studied the effects of a selective leukotriene (LT) antagonist (FPL 57231, 2 mg kg-1 min-1) on the acute cardiopulmonary changes observed in feline endotoxin shock. LTC4 and LTD4 (0.1-3.0 micrograms kg-1) given intravenously had little or no activity on pulmonary arterial pressure (PAP), dynamic lung compliance (Cdyn), and airways resistance (Raw). They did, however, produce a systemic hypertension, which was significantly attenuated during the FPL 57231 infusion. E. coli endotoxin (2 mg kg-1) administration resulted in decreases in systemic arterial blood pressure and Cdyn, together with increases in both PAP and Raw. During infusion of FPL 57231, all these endotoxin-induced cardiopulmonary changes were attenuated. Radioimmunoassay of blood samples taken from cats given FPL 57231 showed that levels of 6-keto prosta-glandin F1 alpha and thromboxane B2 were not significantly increased by endotoxin, as would normally be expected in cats administered endotoxin. FPL 57231 was also found to antagonise the pulmonary effects of the thromboxanemimetic U46619 and of prostaglandin F2 alpha. These results indicate that it is unlikely that the leukotrienes are involved as important mediators of the acute phase of endotoxin shock in cats.     

Adenosine partially prevents cirrhosis induced by carbon tetrachloride in rats

Adenosine administration was tested in rats with carbon tetrachloride-induced hepatic fibrosis and was able to partially prevent the enlargement of liver and spleen induced by the toxin. This amelioration of the hepatomegaly was accompanied by a 50% reduction of the liver collagen deposition and preservation of content of glycosaminoglycans. A stimulated hepatic collagenase activity is apparently the mechanism for reduction of collagen accumulation. These effects were associated with a striking improvement in liver function. Adenosine treatment did not modify the late hepatotoxic effect of the carbon tetrachloride; however, the stimulatory effect of the nucleoside on energy state appeared to counteract the drastic decreases in adenine nucleotides, ATP, ATP/ADP ratio and energy charge elicited by the hepatotoxin. Moreover, a possible beneficial action of enhanced hepatic oxygenation caused by the vasodilator properties of adenosine cannot be ruled out. Regardless of the mechanism, adenosine seems to change the cellular response to the injury induced by the hepatotoxin.     

Nuclear factor-kappaB p50 limits inflammation and prevents lung injury during Escherichia coli pneumonia

Inflammatory responses to infection must be precisely regulated to facilitate microbial killing while limiting host tissue damage. Many inflammatory genes are regulated by kappaB sites, and the p50 subunit of nuclear factor-kappaB suppresses the expression of kappaB-associated genes in vitro. We hypothesized that p50 is essential to prevent excessive inflammation and injury during infection. During pulmonary infection with Escherichia coli, the gene-targeted deficiency of p50 did not affect bacterial clearance from mouse lungs, but it resulted in increased expression of proinflammatory cytokines 6 to 24 hours after infection. This dysregulation exacerbated inflammation (neutrophil recruitment), respiratory distress (pulmonary edema and blood gas exchange impairment), and decompartmentalization (transit of protein and bacteria from the air spaces to the blood). We interpret these studies to indicate that endogenous p50 protects the host by curbing inflammatory responses to prevent injury, essential to survive pneumonia.     

Control of phosphoenolpyruvate-dependent phosphotransferase-mediated sugar transport in Escherichia coli by energization of the cell membrane.

The phosphoenolpyruvate-dependent phosphotransferase-mediated sugar transport in Escherichia coli is inhibited by the energized of the membrane. This was shown in intact cells as well as in membrane vesicles. Relaxation of the proton gradient by uncouplers stimulated the uptake of sugars via the phosphotransferase system in aerobically cultured cells. No such effect was seen in anaerobic cells, apparently because the cell membrane of these cells is poorly energized. Energization by respiration of D-lactate or ascorbate inhibited the phosphotransferase uptake system in membrane vesicles. This inhibition was reversed by the addition of cyanide. Oxamate, a specific inhibitor of lactate dehydrogenase, prevented the inhibitory effect of D-lactate. Membrane vesicles prepared from a cytochrome-less mutant were not energized by D-lactate oxidation and the phosphotransferase uptake system was not inhibited.