Purification and characterization of specific 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase from Escherichia coli B

A phosphatase specific for the hydrolysis of 3-deoxy-d-manno-octulosonate (KDO)-8-phosphate was purified approximately 400-fold from crude extracts of Escherichia coli B. The hydrolysis of KDO-8-phosphate to KDO and inorganic phosphate in crude extracts of E. coli B, grown in phosphate-containing minimal medium, could be accounted for by the enzymatic activity of this specific phosphatase. No other sugar phosphate tested was an alternate substrate or inhibitor of the purified enzyme. KDO-8-phosphate phosphatase was stimulated three- to fourfold by the addition of 1.0 mM Co(+) or Mg(2+) and to a lesser extent by 1.0 mM Ba(2+), Zn(2+), and Mn(2+). The activity was inhibited by the addition of 1.0 mM ethylenediaminetetraacetic acid, Cu(2+), Ca(2+), Cd(2+), Hg(2+), and chloride ions (50% at 0.1 M). The pH optimum was determined to be 5.5 to 6.5 in both tris(hydroxymethyl)aminomethane-acetate and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer. This specific phosphatase had an isoelectric point of 4.7 to 4.8 and a molecular weight of 80,000 +/- 6,000 as determined by molecular sieving and Ferguson analysis. The enzyme appeared to be composed of two identical subunits of 40,000 to 43,000 molecular weight. The apparent K(m) for KDO-8-phosphate was determined to be 5.8 +/- 0.9 x 10(-5) M in the presence of 1.0 mM Co(2+), 9.1 +/- 1 x 10(-5) M in the presence of 1.0 mM Mg(2+), and 1.0 +/- 0.2 x 10(-4) M in the absence of added Co(2+) or Mg(2+).     

Cloning and characterization of the gene encoding 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli.

The cloning of the gene for Escherichia coli PL-2 2-keto-3-deoxy-D-manno-octonate 8-phosphate synthetase is reported. Positive transformants showed an increase of approximately three-fold in specific activity of the enzyme both in E. coli and in Salmonella typhimurium as host cells. A subclone containing a 1.5-kilobase PvuII fragment overproduced active enzyme. Minicell experiments that allow the detection of plasmid encoded proteins revealed an insert-coded single protein band of 34 kilodaltons.     

Escherichia coli YrbI is 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO 8-P) phosphatase, which catalyzes the hydrolysis of KDO 8-P to KDO and inorganic phosphate, is the last enzyme in the KDO biosynthetic pathway for which the gene has not been identified. Wild-type KDO 8-P phosphatase was purified from Escherichia coli B, and the N-terminal amino acid sequence matched a hypothetical protein encoded by the E. coli open reading frame, yrbI. The yrbI gene, which encodes for a protein of 188 amino acids, was cloned, and the gene product was overexpressed in E. coli. The recombinant enzyme is a tetramer and requires a divalent metal cofactor for activity. Optimal enzymatic activity is observed at pH 5.5. The enzyme is highly specific for KDO 8-P with an apparent K(m) of 75 microm and a k(cat) of 175 s(-1) in the presence of 1 mm Mg(2+). Amino acid sequence analysis indicates that KDO 8-P phosphatase is a member of the haloacid dehalogenase hydrolase superfamily.     

Purification and characterization of aminoimidazole ribonucleotide synthetase from Escherichia coli

Aminoimidazole ribonucleotide (AIR) synthetase has been purified 15-fold to apparent homogeneity from Escherichia coli which contains a multicopy plasmid containing the purM, AIR synthetase, gene. The protein is a dimer composed of two identical subunits of Mr 38,500. The N-terminal sequence, amino acid composition, and steady-state kinetics of the protein have been determined. AIR synthetase has been shown to catalyze the transfer of the formyl oxygen of [18O]formylglycinamide ribonucleotide to Pi.     

Higher plants express 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Abstract. The enzymatic activity of 3-deoxy- D-manno -octulosonate 8-phosphate (KDOP) synthase was detected in eight diverse plant species, thus providing enzymological data consistent with recent reports of the presence of 3-deoxy- D-manno -octulosonate in plant cell walls. KDOP synthase from spinach was partially purified and characterized. It possessed weak activity as 3-deoxy- D-arabino -heptulosonate 7-phosphate (DAHP) synthase. In the presence of phosphoenolpyruvate, which conferred dramatic thermostability, KDOP synthase had a catalytic temperature optimum of about 53°C. The pH optimum was 6.2, and divalent cations were neither stimulatory nor required for activity. The Km values for arabinose 5-P and phosphoenolpyruvate were 0.27 mol m⁻³ and about 35 mmol m⁻³, respectively. The kinetics of periodate oxidation of KDOP formed by spinach KDOP synthase indicate that the same stereochemical configuration exists as with bacterial KDOP. The possibility that an unregulated species of DAHP synthase found in some bacteria might in fact be a KDOP synthase exhibiting substrate ambiguity of the type seen in higher plants was examined. However, the DAHP synthase isozyme, DS-O, from Acinetobacter calcoaceticus was found to be specific for erythrose 4-P. The KDOP synthase of Acinetobacter calcoaceticus was also found to be specific for arabinose 5-P.     

Structure and mechanism of 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 A (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (beta/alpha)(8) barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 A apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy.     

Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli.

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.     

Substrate ambiguity of 3-deoxy-D-manno-octulosonate 8-phosphate synthase from Neisseria gonorrhoeae revisited

Homogeneous, recombinant 3-deoxy-D-manno-octulosonate 8-phosphate synthase from Neisseria gonorrhoeae is shown to catalyze the formation of 3-deoxy-D-manno-octulosonate 8-phosphate from phosphoenolpyruvate and D-arabinose 5-phosphate as determined from (1)H-nuclear magnetic resonance analysis of the product. This enzyme does not catalyze the condensation of D-erythrose 4-phosphate and phosphoenolpyruvate to form 3-deoxy-D-ribo-heptulosonate 7-phosphate, as was previously reported (P. S. Subramaniam, G. Xie, T. Xia, and R. A. Jensen, J. Bacteriol. 180:119-127, 1998).     

Purification and characterization of 3-dehydroquinase from Escherichia coli.

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.     

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli.

The phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.2.1.15) was purified to apparent homogeneity from extracts of Escherichia coli K12. The enzyme has a molecular weight of 140,000 as judged by gel filtration and sedimentation equilibrium analysis. The enzyme has a subunit molecular weight of 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native form of the enzyme is a tetramer. This was confirmed by cross-linking the enzyme with dimethylsuberimidate and by analyzing the cross-linked material by gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme shows a narrow pH optimum between pH 6.5 and 7.0. The enzyme is stable for several months when stored at -20 degrees C in buffers containing phosphoenolpyruvate. Removal of phosphoenolpyruvate causes an irreversible inactivation of the enzyme. The enzyme is strongly inhibited by L-phenylalanine and to a lesser degree by dihydrophenylalanine. Molecular parameters of the previously isolated tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from E. coli (Schoner, R., and Herrmann, K.M. (1976) J. Biol. Chem. 251, 5440-5447) are compared with those of the phenylalanine-sensitive isoenzyme from the same organism.     

Purification and immobilization of NAD+ synthetase from Escherichia coli

The enzyme NAD⁺synthetase [deamido-NAD⁺: ammonia ligase (AMP-forming), EC 6.3.1.5] is used for the preparation of 2 μmol isotopically labelled [¹³N]NAD⁺, a radiopharmaceutical designed for positron emission tomography. To obtain a rapid and high yield synthesis of [¹³N]NAD⁺, the NAD⁺synthetase is immobilized on porous glass beads and packed in a column. The NAD⁺synthetase was obtained from Escherichia coli. Different strains were tested; the cell culture technique was optimized. A new, high yield purification was applied. A screening of different immobilization techniques was done. The selected immobilization method was further optimized to increase the enzymatic activity of the enzyme-loaded glass beads. The latter were packed into a glass column. The kinetic properties of this column were investigated and optimized.     

Purification and properties of methionyl-transfer-ribonucleic acid synthetase from Escherichia coli

1. Methionyl-t-RNA synthetase (where t-RNA denotes ;soluble' or transfer RNA) has been purified to apparent homogeneity from a ribonuclease I-free strain of Escherichia coli. Polyacrylamide-gel electrophoresis of the final product revealed a single band. The purified enzyme catalyses the exchange of 450mumoles of pyrophosphate into ATP/mg. in 15min. at 37 degrees . 2. Methionyl-t-RNA synthetase is specific for the l-isomer of methionine, but appears to catalyse the methionylation of two distinct species of t-RNA, both of which are specific for methionine, but only one of which may be subsequently formylated. 3. The Michaelis constant for l-methionine is 2x10(-4)m in the ATP-PP(i) exchange assay and 2x10(-5)m for the acylation of t-RNA. 4. Gel filtration of both crude and highly purified preparations of methionyl-t-RNA synthetase on Sephadex G-200 indicates that the active species of enzyme has a molecular weight of about 190000. The amino acid composition of the enzyme is similar to those reported for the isoleucine and tyrosine enzymes from E. coli.     

Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The aroH gene of Escherichia coli, which encodes the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzyme of the common aromatic biosynthetic pathway, was cloned behind the tac promoter in expression plasmid pKK223-3. The enzyme was overexpressed, purified to homogeneity, and characterized. The native enzyme was found to be a dimeric metalloprotein containing 0.3 mol of iron per mol of subunit and variable amounts of zinc. The activity of the native enzyme was stimulated two- to threefold when assayed in the presence of Fe2+ ions. Pretreatment of the enzyme with Fe2+ also resulted in activation, accompanied by an equivalent increase in iron content. Treatment of the enzyme with chelating agents led to inactivation, which was fully reversed by the presence of Fe2+ in the assay mixture. The native enzyme exhibited a unique absorption profile, having a shoulder of absorbance on the aromatic band with a maximum around 350 nm and a broad, weak band with a maximum around 500 nm. Treatment of the enzyme with Fe2+ enhanced the absorbance at 350 nm and eliminated the band at 500 nm. Treatment with reducing agents caused the disappearance of both bands and destabilized the enzyme. Feedback regulation of the activity of the enzyme was specific for tryptophan, with maximum inhibition at about 70%.     

Purification and positional specificity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli membranes.

SN-Glycerol-3-phosphate acyltransferase was solubilized from membranes of Escherichia coli B and K-12 and purified on an affinity column of Sepharose 4B coupled with 6-phosphogluconic acid. Phosphatidylglycerol was required for activation and stabilization of the purified enzyme. The acyl residues were exclusively transferred to the position 1 of sn-glycerol 3-phosphate by the enzyme, regardless of whether the acyl-CoA was saturated or unsaturated.     

Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli

Most parasitic protozoa lack the de novo purine biosynthetic pathway and rely exclusively on the salvage pathway for their purine nucleotide requirements. Enzymes of the salvage pathway are, therefore, candidate drug targets. We have cloned the Plasmodium falciparum adenylosuccinate synthetase gene. In the parasite, adenylosuccinate synthetase is involved in the synthesis of AMP from IMP formed during the salvage of the purine base, hypoxanthine. The gene was shown to code for a functionally active protein by functional complementation in a purA mutant strain of Escherichia coli, H1238. This paper reports the conditions for hyperexpression of the recombinant protein in E. coli BL21(DE3) and purification of the protein to homogeneity. The enzyme was found to require the presence of dithiothreitol during the entire course of the purification for activity. Glycerol and EDTA were found to stabilize enzyme activity during storage. The specific activity of the purified protein was 1143.6 +/- 36.8 mUnits/mg. The K(M)s for the three substrates, GTP, IMP, and aspartate, were found to be 4.8 microM, 22.8 microM, and 1.4 mM, respectively. The enzyme was a dimer on gel filtration in buffers of low ionic strength but equilibrated between a monomer and a dimer in buffers of increased ionic strength.