The Involvement of Hydrogenases 1 and 2 in the Hydrogen-Dependent Nitrate Respiration of Escherichia coli

The study of Escherichia coli mutants synthesizing either hydrogenase 1 (HDK203) or hydrogenase 2 (HDK103) showed that the nitrate-dependent uptake of hydrogen by E. coli cells can be accomplished through the action of either of these hydrogenases. The capability of the cells for hydrogen-dependent nitrate respiration was found to depend on the growth conditions. E. coli cells grown anaerobically without nitrate in the presence of glucose were potentially capable of nitrate-dependent hydrogen consumption. The cells grown anaerobically in the presence of nitrate exhibited a much lower capability for nitrate-dependent hydrogen consumption. The inhibitory effect of nitrate on this capability of bacterial cells was either weak (the mutant HDK203) or almost absent (the mutant HDK103) when the cells were grown in the presence of peptone and hydrogen. Hydrogen stimulated the growth of the wild-type strain and the mutant HDK103 (but not the mutant HDK203) cultivated in the medium with nitrate and peptone. These data suggest that hydrogenase 2 is much more active in catalyzing nitrate-dependent hydrogen consumption than hydrogenase 1.     

Purification and characterization of reaction centers from the obligate aerobic phototrophic bacteria Erythrobacter litoralis,Erythromonas ursincola and Sandaracinobacter sibiricus

Reaction centers (RC) from the species Erythrobacter (Eb.) litoralis, Erythromonas (Em.) ursincola and Sandaracinobacter (S.) sibiricus have been purified by LDAO treatment of light-harvesting-reaction center complexes and DEAE chromatography. The content and overall organisation of the RCs' chromophores, determined by linear dichroism (LD) and absorption spectroscopy, are similar to those isolated from anaerobic photosynthetic bacteria. The redox properties of the primary electron donor are pH-independent and very similar to those determined for anaerobic photosynthetic bacteria with midpoint potential values equal to 445 (± 10), 475 and 510 mV for Eb. litoralis, S. sibiricus and Em. ursincola, respectively. The RC purified from Eb. litoralis does not contain bound cytochrome (cyt), whereas RCs isolated from S. sibiricus and Em. ursincola possess a tetraheme cyt c. Each of these tetraheme cyts contains two high potential hemes and two low potential hemes. Their redox properties are very similar, with midpoint potentials equal to 385 (± 10), 305, 40, -40 mV for Em. ursincola and 355, 285, 30, -48 mV for S. sibiricus. At physiological pH, the midpoint potential of the primary electron acceptor (Q_A) varies with a slope of -60 mV/pH unit. The reduced form of Q_A presents pK values of 9, 9.8, 10.5 for S. sibiricus, Em. ursincola and Eb. litoralis, respectively. The main difference observed between RCs isolated from anaerobic photosynthetic and from obligate aerobic bacteria is the _Emvalues of Q_A which are 65 to 120 mV higher in the last case. This difference is proposed to be a major reason for the inability of these species to grow under anaerobic photosynthetic conditions.     

Purification and properties of N-acetylneuraminate lyase from Escherichia coli

N-Acetylneuraminate lyase [N-acetylneuraminic acid aldolase EC 4.1.3.3] from Escherichia coli was purified by protamine sulfate treatment, fractionation with ammonium sulfate, column chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA 44, and preparative polyacrylamide gel electrophoresis. The purified enzyme preparation was homogeneous on analytical polyacrylamide gel electrophoresis, and was free from contaminating enzymes including NADH oxidase and NADH dehydrogenase. The enzyme catalyzed the cleavage of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate in a reversible reaction. Both cleavage and synthesis of N-acetylneuraminic acid had the same pH optimum around 7.7. The enzyme was stable between pH 6.0 to 9.0, and was thermostable up to 60 degrees C. The thermal stability increased up to 75 degrees C in the presence of pyruvate. No metal ion was required for the enzyme activity, but heavy metal ions such as Ag+ and Hg2+ were potent inhibitors. Oxidizing agents such as N-bromosuccinimide, iodine, and hydrogen peroxide, and SH-inhibitors such as p-chloromercuribenzoic acid and mercuric chloride were also potent inhibitors. The Km values for N-acetylneuraminic acid and N-glycolylneuraminic acid were 3.6 mM and 4.3 mM, respectively. Pyruvate inhibited the cleavage reaction competitively; Ki was calculated to be 1.0 mM. In the condensation reaction, N-acetylglucosamine, N-acetylgalactosamine, glucosamine, and galactosamine could not replace N-acetylmannosamine as substrate, and phosphoenolpyruvate, lactate, beta-hydroxypyruvate, and other pyruvate derivatives could not replace pyruvate as substrate. The molecular weight of the native enzyme was estimated to be 98,000 by gel filtration methods. After denaturation in sodium dodecyl sulfate or in 6 M guanidine-HCl, the molecular weight was reduced to 33,000, indicating the existence of 3 identical subunits. The enzyme could be used for the enzymatic determination of sialic acid; reaction conditions were devised for determining the bound form of sialic acid by coupling neuraminidase from Arthrobacter ureafaciens, lactate dehydrogenase, and NADH.     

Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli.

beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5. The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM. The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000. It is shown to be a soluble cytoplasmic enzyme. Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase.     

Purification and properties of diaminopimelate decarboxylase from Escherichia coli

1. Diaminopimelate decarboxylase from a soluble extract of Escherichia coli A.T.C.C. 9637 was purified 200-fold by precipitation of nucleic acids, fractionation with acetone and then with ammonium sulphate, adsorption on calcium phosphate gel and chromatography on DEAE-cellulose or DEAE-Sephadex. 2. The purified enzyme showed only one component in the ultracentrifuge, with a sedimentation coefficient of 5·4s. One major peak and three much smaller peaks were observed on electrophoresis of the enzyme at pH8·9. 3. The mol.wt. of the enzyme was approx. 200000. The catalytic constant was 2000mol. of meso-diaminopimelic acid decomposed/min./mol. of enzyme, at 37°. The relative rates of decarboxylation at 25°, 37° and 45° were 0·17:1·0:1·6. At 37° the Michaelis constant was 1·7mm and the optimum pH was 6·7–6·8. 4. There was an excess of acidic amino acids over basic amino acids in the enzyme, which was bound only on basic cellulose derivatives at pH6·8. 5. The enzyme had an absolute requirement for pyridoxal phosphate as a cofactor; no other derivative of pyridoxine had activity. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was also needed as an activator. 6. In the presence of 2,3-dimercaptopropan-1-ol (1mm), heavy-metal ions (Cu²⁺, Hg²⁺) did not inhibit the enzyme, but there was inhibition by several amino acids with analogous structures to diaminopimelate, generally at high concentrations relative to the substrate. Penicillamine was inhibitory at relatively low concentrations; its action was prevented by pyridoxal phosphate.     

Purification and properties of hydroxymethylpyrimidine kinase from Escherichia coli

Hydroxymethylpyrimidine kinase, which catalyzes the conversion of 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) to its monophosphate, is purified about 3300-fold to apparent homogeneity from the cell-free extracts of E. coli K-12 through four successive steps of column chromatographies. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis and its molecular weight is estimated to be 43 000-44 000. The enzyme phosphorylated each of the pyridoxine substrates, pyridoxine, pyridoxal and pyridoxamine as well as hydroxymethylpyrimidine, and the reaction gave rise to a corresponding 5'-phosphate compound. The Km values of the purified enzyme for hydroxymethylpyrimidine and for pyridoxine are 1.1.10(-4) and 6.6.10(-5) M, respectively. Pyridoxine inhibits competitively the phosphorylation of hydroxymethylpyrimidine with a Ki value of 2.7.10(-6) M and hydroxymethylpyrimidine shows the same for that of pyridoxine with a Ki value of 9.0.10(-5) M. A similarity in enzymic properties between the hydroxymethylpyrimidine kinase and an enzyme which has been characterized as pyridoxal kinase leads to the assumption that both hydroxymethylpyrimidine and pyridoxine might be phosphorylated by the same enzyme species.     

Purification and properties of citrate lyase from Escherichia coli

Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction. The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis. The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons. An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones. The polypeptide subunits have been isolated in pure state and their biological functions characterize. The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid. The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A. The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits. The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases. A ligase which acetylates the deacetyl[citrate lyase] in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS)