Phosphorylation of isocitrate lyase in Escherichia coli

Isocitrate lyase from Escherichia coli becomes phosphorylated in vitro by an endogenous kinase when partially purified extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with histidine modifying reagents, and alkaline hydrolysis of in vitro phosphorylated enzyme indicated the presence of a phosphohistidine residue. Phosphorylation of isocitrate lyase can also occur in vivo, which indicates a possible regulatory significance of this modification. In addition to phosphorylation, isocitrate lyase is capable of incorporating label from both [alpha-32P]ATP and [14C]ATP suggesting that more than one type of covalent modification occurs on this enzyme. This report reviews the studies which have demonstrated the phosphorylation and modification of isocitrate lyase from Escherichia coli.     

Phosphorylation of an Escherichia coli protein at tyrosine

The analysis of protein phosphorylation in the bacterium Escherichia coli showed that, while most phosphoproteins are modified at serine and/or threonine residues, one of them is modified exclusively at tyrosine. This particular protein which has a molecular weight of 54,500 and a pHi value of 5.6 is found associated with the membrane/ribosome fraction of the cell.     

Membrane Translocation of Mannitol in Escherichia coli Without Phosphorylation

Galactosyl-mannitol can be transported into cells of Escherichia coli by beta-galactoside permease and can be hydrolyzed rapidly to mannitol and galactose by beta-galactosidase. When a mutant strain lacking enzyme I of the phosphoenolpyruvate phosphotransferase system and constitutive in the lactose system was presented with galactosyl-mannitol in which the mannitol moiety was labeled with (3)H, the liberated mannitol remained inside the cell if the Enzyme II complex of the phosphoenolpyruvate phosphotransferase system for mannitol was uninduced. It is postualted that one of the enzyme II proteins can still catalyze translocation of mannitol across the cell membrane even when phsophorylation is not possible.     

Phosphorylation of Escherichia coli proteins during the SOS response

• 1.1. The phosphorylation of Escherichia coli proteins was analyzed comparatively before and after induction of the SOS response in a temperature-sensitive mutant strain.
• 2.2. The presence of phosphorylated proteins was evidenced by gel electrophoresis and autoradiography after labelling with radioactive orthophosphate in vivo or radioactive adenosine triphosphate in vitro.
• 3.3. Significant changes in the intensity of protein labelling were observed upon induction of the SOS functions: six proteins were found to be more phosphorylated while two others were less phosphorylated. Moreover, five additional proteins appeared to become phosphorylated exclusively during the SOS response. The molecular mass and isoelectric point of these various proteins were determined.
• 4.4. For most proteins, the changes in the pattern of protein phosphorylation were concomitant with variations in the amount of protein synthesized.
• 5.5. The changes in the pattern of phosphoproteins observed during the SOS response were not due to the temperature shift required experimentally for expressing the SOS phenotype.
• 6.6. Phosphorylation was found to be catalyzed by protein kinases that modify amino acid residues at hydroxyl groups in protein substrates.
• 7.7. Both in vivo and in vitro studies brought evidence that neither RecA nor LexA, the two key regulatory proteins of the SOS functions, were capable of undergoing phosphorylation.

     

Crystal structure of the Escherichia coli RNA degradosome component enolase.

The crystal structure of Escherichia coli enolase (EC 4.2.1.11, phosphopyruvate hydratase), which is a component of the RNA degradosome, has been determined at 2.5 A. There are four molecules in the asymmetric unit of the C2 cell, and in one of the molecules, flexible loops close onto the active site. This closure mimics the conformation of the substrate-bound intermediate. A comparison of the structure of the E. coli enolase with the eukaryotic enolase structures available (lobster and yeast) indicates a high degree of conservation of the hydrophobic core and the subunit interface of this homodimeric enzyme. The dimer interface is enriched in charged residues compared with other protein homodimers, which may explain our observations from analytical ultracentrifugation that dimerisation is affected by ionic strength. The putative role of enolase in the RNA degradosome is discussed; although it was not possible to ascribe a specific role to it, a structural role is possible.     

Phosphorylation of 1-deoxy-D-xylulose by D-xylulokinase of Escherichia coli.

1-deoxy-D-xylulose 5-phosphate serves as a precursor for the biosynthesis of the vitamins thiamine and pyridoxal and for the formation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate via the nonmevalonate pathway of terpenoid biosynthesis. Earlier studies had shown that Escherichia coli incorporates unphosphorylated 1-deoxy-D-xylulose into the terpenoid side chain of ubiquinones with high efficacy. We show that D-xylulokinase of E. coli (EC 2.7.1.17) catalyzes the phosphorylation of 1-deoxy-D-xylulose at the hydroxy group of C-5 at a rate of 1.6 micromol.mg min-1. This reaction constitutes a potential salvage pathway for the generation of 1-deoxy-D-xylulose 5-phosphate from exogenous or endogenous 1-deoxy-D-xylulose as starting material for the biosynthesis of terpenoids, thiamine and pyridoxal.     

Uncoupling of Substrate-Level Phosphorylation in Escherichia coli during Glucose-Limited Growth

The respiratory chain of Escherichia coli contains three different cytochrome oxidases. Whereas the cytochrome bo oxidase and the cytochrome bd-I oxidase are well characterized and have been shown to contribute to proton translocation, physiological data suggested a nonelectrogenic functioning of the cytochrome bd-II oxidase. Recently, however, this view was challenged by an in vitro biochemical analysis that showed that the activity of cytochrome bd-II oxidase does contribute to proton translocation with an H(+)/e(-) stoichiometry of 1. Here, we propose that this apparent discrepancy is due to the activities of two alternative catabolic pathways: the pyruvate oxidase pathway for acetate production and a pathway with methylglyoxal as an intermediate for the production of lactate. The ATP yields of these pathways are lower than those of the pathways that have so far always been assumed to catalyze the main catabolic flux under energy-limited growth conditions (i.e., pyruvate dehydrogenase and lactate dehydrogenase). Inclusion of these alternative pathways in the flux analysis of growing E. coli strains for the calculation of the catabolic ATP synthesis rate indicates an electrogenic function of the cytochrome bd-II oxidase, compatible with an H(+)/e(-) ratio of 1. This analysis shows for the first time the extent of bypassing of substrate-level phosphorylation in E. coli under energy-limited growth conditions.     

Phosphorylation inactivates Escherichia coli isocitrate dehydrogenase by preventing isocitrate binding

Equilibrium binding studies demonstrate that purified Escherichia coli isocitrate dehydrogenase binds isocitrate, alpha-ketoglutarate, NADP, and NADPH at 1:1 ratios of substrate to enzyme monomer. The phosphorylated enzyme, which is completely inactive, is unable to bind isocitrate but retains the ability to bind NADP and NADPH. Replacement of serine 113, which is the site of phosphorylation, by aspartate results in an inactive enzyme that is unable to bind isocitrate. Replacement of the same serine with other amino acids (lysine, threonine, cysteine, tyrosine, and alanine) produces active enzymes that bind both substrates. Hence, the negative charge of an aspartate or a phosphorylated serine at site 113 inactivates the enzyme by preventing the binding of isocitrate.     

Expression of DNA sequences containing neuron specific enolase gene in Escherichia coli.

There is evidence that the gene for gamma-gamma enolase (neuron specific enolase, NSE) is regulated during cell differentiation and development, conserved in a variety of organisms and contains mRNA destabilizing sequences. In order to investigate further the mechanisms of these processes and to obtain large quantity of this protein, the NSE gene was isolated from neuroblastoma cells and cloned in E. coli using standard molecular biology techniques. The NSE gene expression was studied and the expressed protein (recombinant NSE) was characterized extensively. The recombinant NSE behaves like parental NSE in antisera specificity, resistance for chaotropic agents like urea, thermal stability at higher temperatures etc. The physical parameters like secondary structure, hydrophilicity, antigenic index and flexibility of the expressed protein were studied. The results of the present investigation collectively form the basis for initial investigations of how the expression of NSE gene is regulated. This is the first report where the recombinant NSE gene has been characterized so extensively.     

Induction by Oxygen of Respiration and Phosphorylation of Anaerobically Grown Escherichia coli

The changes occurring in the respiratory enzymes of anaerobically grown Escherichia coli strain B and E. coli 15 T⁻A⁻U⁻bar during exposure to oxygen were studied. Reduced nicotinamide adenine dinucleotide (NADH) oxidase activity reached its peak soon after O₂ exposure; cytochrome content and succinate oxidase activity increased more slowly, and these increases paralleled each other. The activities of isocitrate and malate dehydrogenases also increased, but the increase was less than that of the succinate and NADH oxidases; exposure to O₂ had no effect on the succinate and NADH dehydrogenase activities. On the other hand, the glycolytic activity decreased slowly after O₂ exposure. The incorporation of ³²P into acid-soluble organic phosphate esters paralleled the respiratory rate during the first 60 min after O₂ exposure, but continued to increase after the respiration reached a plateau. The sensitivity of ³²P incorporation to the uncoupler carbonyl cyanide m-chlorophenylhydrazone also increased with time. The observed relationship between the development of the respiratory chain and the energy-conserving mechanism during O₂ exposure is discussed. Synthesis of the respiratory enzymes upon exposure to oxygen was dependent on concomitant protein and ribonucleic acid synthesis but not on deoxyribonucleic acid synthesis.     

Phosphorylation of Escherichia coli RNA polymerase by rabbit skeletal muscle protein kinase

Rabbit skeletal muscle protein kinase catalyzes the phosphorylation of DNA-dependent RNA polymerase of Escherichia coli in the presence of adenosine 3′,5′-monophosphate and ATP. The phosphorylation occurs on one (or more) serine residue(s) in the σ-factor under reaction conditions similar to those employed for RNA synthesis. The phosphorylation of RNA polymerase and its stimulation by protein kinase are inhibited by a specific heat-stable inhibitor from rabbit skeletal muscle. With conditions more favorable for the protein kinase reaction, phosphorylation of RNA polymerase also occurs on the β subunit of the core enzyme, but this reaction occurs at a much slower rate than the phosphorylation of the σ-factor.     

RNase G-dependent degradation of the eno mRNA encoding a glycolysis enzyme enolase in Escherichia coli

Escherichia coli RNase G, encoded by the rng gene, is involved in the processing of 16S rRNA and degradation of the adhE mRNA encoding a fermentative alcohol dehydrogenase. In a search for the intracellular target RNAs of RNase G other than the 16S rRNA precursor and adhE mRNA, total cellular proteins from rng+ and rng::cat cells were compared by two-dimensional gel electrophoresis. The amount of enolase encoded by the eno gene reproducibly increased two- to three-fold in the rng::cat mutant strain compared with the rng+ parent strain. Rifampicin chase experiments showed that the half-life of the eno mRNA was some 3 times longer in the rng::cat mutant than in the wild type. These results indicate that the eno mRNA was a substrate of RNase G in vivo, in addition to 16S rRNA precursor and adhE mRNA.     

Phosphorylation due to the oxidation of succinic acid by cell-free extracts of Escherichia coli

Esterification of inorganic phosphate accompanies the oxidation of succinic acid by cell-free extracts of E. coli. Inorganic phosphate appears to be an essential requirement for the dehydrogenation of the C₄-dicarboxylic acid.     

Phosphorylation of adenosine 5'-monophosphate by a dialyzed cell-free extract from Escherichia coli.

A cell-free extract of Escherichia coli, even after exhaustive dialysis, was found capable of phosphorylating adenosine 5'-monophosphate (AMP) to adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP). Centrifugation at 100 000 g for 3h sedimented most of the capacity to phosphorylate AMP to ATP, while the supernatant retained a significant capacity to phosphorylate AMP to ADP. The pellet contained a greater amount of phosphate polymers (which were neither DNA, RNA, nor proteins) than did the supernatant. The addition of authentic inorganic polyphosphates to the supernatant restored the phosphorylating capacity of the original extracts. It is concluded that the observed phosphorylation is partly due to inorganic polyphosphate.     

Purification and characterization of enolase fromEscherichia coli

A method for the purification of enolase (EC 4.2.1.11) from an overproducing strain ofEscherichia coli JA 200 pLC 11–8 is described. The procedure included treatment of the crude sonic extract with protamine sulfate, followed by ammonium sulfate fractionation, hydrophobic interaction chromatography with phenyl Sepharose, HPLC ion exchange chromatography with a DuPont Sax column, and HPLC hydrophobic interaction chromatography with a Bio-Rad 5-PW column. The enzyme was purified to homogeneity as determined by silver staining of 10% sodium dodecylsulfate polyacrylamide gels. The native molecular weight ofE. coli enolase was found to be 92 kilodaltons and consisted of two subunits of identical molecular weight, 46 kilodaltons each. The isoelectric point was found to be 4.9.