Reversible desensitization of phosphoenolpyruvate carboxylase to multiple effectors by butanedione.

Chemical modification of phosphoenolpyruvate carboxylase [EC 4.1.1.31] of Escherichia coli W with 2,3-butanedione, an arginyl residue reagent, results in an inactivation of the enzyme. The inactivation proceeds following pseudo-first order kinetics. DL-Phospholactate, a substrate analog, effectively protects the enzyme from the inactivation. The enzyme modified in the presence of DL-phospholactate or in its absence is completely desensitized to fructose 1,6-bisphosphate and GTP, allosteric activators for the enzyme. At the same time, the sensitivities to acetyl coenzyme a, laurate and L-aspartate are considerably decreased. Resensitization is attained, however, upon removal of excess butanedione and borate by gel filtration, concomitant with the restoration of the catalytic activity.     

Protein synthesis results in guanosine-5'-diphosphate-3'-diphosphate synthesis in Escherichia coli minicells.

Minicells of Escherichia coli DS410 synthesized guanosine-5'-diphosphate-3'-diphosphate and guanosine-5'-triphosphate-3'-diphosphate when synthesizing proteins in response to phage T7 infection. The guanosine-5'-diphosphate-3'-diphosphate synthesis was found to be relA+ dependent and inhibited by chloramphenicol.     

The effect of alcohols on guanosine 5'-diphosphate-3'-diphosphate metabolism in stringent and relaxed Escherichia coli

The effects of a series of alcohols on the stringent response system of Escherichia coli were studied. The alcohols used could be divided into two groups on the basis of the response of pppGpp and ppGpp to the growth downshift induced by the alcohols. The cells responded to the alcohols, methanol, ethanol, and propanol, as if they were being starved of amino acids. In the stringent strain CP78 these alcohols induced pppGpp and ppGpp accumulation and curtailed RNA synthesis, whereas in the relaxed strain CP79, both of these responses were absent. It was determined that this response was most likely due to an interference by these alcohols with the uptake of amino acids required by these strains. By contrast both stringent and relaxed cells elevated their level of ppGpp and decreased RNA accumulation when treated with butanol or pentanol. This response is similar to the effect of carbon source limitation. It was determined that the elevation of ppGpp in the stringent strain was primarily the result of increased ppGpp synthesis in response to these alcohols. In the relaxed strain the rise in ppGpp was dependent on a decrease in ppGpp degradation coupled with a moderate increase in ppGpp synthesis. This stimulation of ppGpp synthesis in relaxed cells, although small, suggests the existence of an enzyme distinct from stringent factor which is capable of synthesizing ppGpp. Data are presented which suggest that the activity of this enzyme is coupled to the potential for protein synthesis and energy availability of the cell, perhaps being regulated by the overall ratio of unchanged to amino-acylated tRNA.     

The Escherichia coli adenylate cyclase complex: Activation by phosphoenolpyruvate

A model for the regulation of the activity of Escherichia coli adenylate cyclase is presented. It is proposed that Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) interacts in a regulatory sense with the catalytic unit of adenylate cyclase. The phosphoenolpyruvate (PEP)-dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state of adenylate cyclase. The pyruvate or sugar-dependent dephosphorylation of Enzyme I is correlated with a low activity state of adenylate cyclase. Evidence in support of the proposed model involves the observation that Enzyme I mutants have low cAMP levels and that PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase, Kinetic studies indicate that various ligands have opposing effects on adenylate cyclase. While PEP activates the enzyme, either glucose or pyruvate inhibit it. The unique relationships of PEP and Enzyme I to adenylate cyclase activity are discussed.     

Activation of transcription by guanosine 5'-diphosphate,3'-diphosphate, transfer ribonucleic acid, and novel protein from Escherichia coli.

A protein factor TFms) that is required for ppGpp to stimulate RNA synthesis has been purified from an eluate of crude ribosomes. TFms also has the capacity to stimulate RNA synthesis without ppGpp present. Under standard conditions the action TFms and ppGpp requires uncharged tRNA. TFms and ppGpp act at inhibition to promote the formation of rifampicin-resistant or polytrI)-resistant preinitiation complexes. In the presence of rifampicin or poly(rI), tRNA is no longer required. With lambdah80dlacPs DNA as template, ppGpp together with TFms stimulated gal RNA synthesis to a much greater extent than total RNA synthesis. The stimulation of both lac and gel RNA synthesis was increased in the presence of cyclic AMP receptor and cyclic AMP.     

Inhibition of maize leaf phosphoenolpyruvate carboxylase by diethyl oxaloacetate

Diethyl oxaloacetate was found to be a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase activity with respect to the substrate phosphoenolpyruvate. The K_i values, based on total diethyl oxaloacetate, decreased with increasing pH, while the K_i values, based on the enol tautomer (average of 4 M), were similar and independent of pH. The results suggest that inhibition is dependent on the enol tautomer. Diethyl oxaloacetate was a weak inhibitor following treatment of the enzyme with dithiothreitol; inhibition could be restored by treatment with diamide, indicating inhibition depends on the reduction state of thiol groups on the enzyme.Abbreviations DTT dithiothreitol - HPLC high performance liquid chromatography - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - Tricine N-tris(hydroxymethyl)methylglycine     

Characterization of the guanosine 5'-triphosphate 3'-diphosphate and guanosine 5'-diphosphate 3'-diphosphate degradation reaction catalyzed by a specific pyrophosphorylase from Escherichia coli.

Guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp) are specifically degraded by a manganese-dependent pyrophosphorylase present in spoT+ but not in spoT- strains of Escherichia coli, indicating that the enzyme is the spoT gene product. The enzyme catalyzes the release of pyrophosphate from the 3' position of ppGpp or pppGpp, yielding ppG and pppG, respectively; pppGpp could not be detected as an intermediate in the decay reaction. Degradation of (p)ppGpp is optimal in the presence of 200 to 300 mM potassium or sodium acetate, at a pH of 7.5 to 8 and a temperature of 37 degrees C.     

Synthesis of guanosine 5'-diphosphate, 3'-diphosphate in spot mutants of Escherichia coli.

The synthesis of ppGpp in spoT- mutants of Escherichia coli has been invesitgated. In these mutants the first-order rate constant for ppGpp breakdown is low, and pppGpp is barely detectable. It is shown that the rate of pppGpp, and hence ppGpp, synthesis is strongly reduced compared with that observed in spot+ strains. The low rate of magic spot synthesis satisfactorily explains the low levels of pppGpp in spoT- mutants. The pentaphosphate very probably is the precursor of ppGpp as it is in wild-type, i.e. spoT+, strains.     

Degradation of guanosine 3'-diphosphate 5'-diphosphate in vitro by the spoT gene product of Escherichia coli.

Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) is rapidly degraded to guanosine 5'-diphosphate (ppG) and probably pyrophosphate by an enzyme present in the ribosomal fraction prepared from spoT+ strains of Escherichia coli. The ppGpp-degrading enzyme was released from the ribosomes during dissociation at low ionic strength. Ribosomes are not essential for degradation of ppGpp, and decay of ppGpp is strictly dependent on manganese ions. The reaction is sensitive to inhibition by tetracycline, which can be reversed by MnCl2, indicating that the inhibitory effect is due to the previously described chelating properties of the antibiotic. When the ppGpp-degrading enzyme was complemented with adenosine 5'-triphosphate (pppA) and a nucleoside diphosphate kinase, decay of ppGpp was accelerated yielding pppG and ppG as major products. In the absence of pppA we have been unable to detect the ppGpp-degrading enzyme in various spoT- mutant strains indicating that this enzyme is the spoT gene product.     

Alteration of growth yield by overexpression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in Escherichia coli.

Phosphoenolpyruvate and oxaloacetate are key intermediates at the junction between catabolism and biosynthesis. Alteration of carbon flow at these branch points will affect the growth yield and the formation of products. We attempted to modulate the metabolic flow between phosphoenolpyruvate and oxaloacetate by overexpressing phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase from a multicopy plasmid under the control of the tac promoter. It was found that overexpression of phosphoenolpyruvate carboxylase decreased the rates of glucose consumption and organic acid excretion, but the growth and respiration rates remained unchanged. Consequently, the growth yield on glucose was improved. This result indicates that the wild-type level of phosphoenolpyruvate carboxylase is not optimal for the most efficient glucose utilization in batch cultures. On the other hand, overexpression of phosphoenolpyruvate carboxykinase increased glucose consumption and decreased oxygen consumption relative to those levels required for growth. Therefore, the growth yield on glucose was reduced because of a higher rate of fermentation product excretion. These data provide useful insights into the regulation of central metabolism and facilitate further manipulation of pathways for metabolite production.     

Molecular basis for the glyphosate-insensitivity of the reaction of 5-enolpyruvylshikimate 3-phosphate synthase with shikimate

The shikimate pathway enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSP synthase) has received attention in the past because it is the target of the broad-spectrum herbicide glyphosate. The natural substrate of EPSP synthase is shikimate-3-phosphate. However, this enzyme can also utilize shikimate as substrate. Remarkably, this reaction is insensitive to inhibition by glyphosate. Crystallographic analysis of EPSP synthase from Escherichia coli, in complex with shikimate/glyphosate at 1.5 Angstroms resolution, revealed that binding of shikimate induces changes around the backbone of the active site, which in turn impact the efficient binding of glyphosate. The implications from these findings with respect to the design of novel glyphosate-insensitive EPSP synthase enzymes are discussed.