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.     

Temperature-sensitive mutation affecting synthesis of phosphoenolpyruvate carboxykinase in Escherichia coli.

A mutation has been characterized in Escherichia coli which results in temperature-sensitive expression of phosphoenolpyruvate carboxykinase activity and antigen. The enzyme produced by the mutant strain at a permissive temperature or by cells treated with chloramphenicol at nonpermissive temperatures had normal activity and stability in extracts. Since phosphoenolpyruvate carboxykinase had a monomeric structure, the mutation probably affects the synthesis, rather than the structure or assembly, of the enzyme.     

A mutant phosphoenolpyruvate carboxykinase in Escherichia coli conferring oxaloacetate decarboxylase activity.

The phosphoenolpyruvate carboxykinase in Escherichia coli (encoded by pck) catalyzes the conversion from oxaloacetate (OAA) to phosphoenolpyruvate under gluconeogenic conditions. We report here the characterization of two mutant alleles, pck-51 and pck-53, both of which are point mutations leading to single amino acid changes (D to N at position 268 and G to S at position 284, respectively). Pck51 is an altered-activity mutant that catalyzes the conversion from OAA to pyruvate (OAA decarboxylase activity). This new activity was not detected from the wild-type Pck, and it complements the pck null mutation only in a pps+ background. Pck53 is a reduced-activity mutant that complements the pck null mutation in a strain-dependent fashion.     

Crystallization of the calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli K12

Single crystals of phosphoenolpyruvate carboxykinase from Escherichia coli K12 have been grown in the orthorhombic crystal system. Single crystals developed to a maximum size of 0.25 mm x 0.25 mm x 1.5 mm by the technique of washing and reseeding. The space group is P2(1)2(1)2(1), with a = 77.24 A, b = 89.18 A, c = 93.24 A and Z = 4; there is one enzyme molecule per crystallographic asymmetric unit and the solvent content is estimated to be 59%. The crystals diffract to at least 2.8 A d spacings and decompose in the X-ray beam after approximately two days of exposure.     

On the monomeric structure and proposed regulatory properties of phosphoenolpyruvate carboxykinase of Escherichia coli.

Phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating)) (EC 4.1.1.49) has been purified to homogeneity from Escherichia coli. The enzyme shows the same molecular weight (ca. 65000) either by sedimentation equilibrium under nondenaturing conditions or by polyacrylamide gel electrophoresis in the presence of detergent, indicating that the enzyme has a monomeric structure. We have confirmed the previous observation that NADH is an inhibitor of this enzyme, but we have failed to detect the previously reported appearance of homotropic cooperativity with respect to substrate binding the presence of this inhibitor. Lack of such homotropic interactions is in harmony with our conclusion that the enzymes is a monomer. Replacement of Mg2+ by Mn2+ in the assay medium lowers the Km for phosphoenolpyruvate by an order of magnitude, but does not affect the characteristics of inhibition by NADH.     

Substrate-induced conformational changes of melibiose permease from Escherichia coli studied by infrared difference spectroscopy

Fourier transform infrared difference spectroscopy has been used to obtain information about substrate-induced structural changes of the melibiose permease (MelB) from Escherichia coli reconstituted into liposomes. Binding of the cosubstrate Na(+) gives rise to several peaks in the amide I and II regions of the difference spectrum Na(+).MelB minus H(+).MelB, that denote the presence of conformational changes in all types of secondary structures (alpha-helices, beta-sheets, loops). In addition, peaks around 1400 and at 1740-1720 cm(-1) are indicative of changes in protonation/deprotonation or in environment of carboxylic groups. Binding of the cosubstrate Li(+) produces a difference spectrum that is also indicative of conformational changes, but that is at variance as compared to that induced by Na(+) binding. To analyze the following transport steps, the melibiose permease with either H(+), Na(+), or Li(+) bound was incubated with melibiose. The difference spectra obtained by subtracting the spectrum cation.MelB from the respective complex cation.melibiose.MelB were roughly similar among them, but different from those induced by cation binding, and more intense. Therefore, major conformational changes that are induced during melibiose binding/substrate translocation, like those denoted by intense peaks at 1668 and 1645 cm(-)(1), are similar for the three cotransporting cations. Changes in the protonation state and/or in the environment of given carboxylic residues were also induced by melibiose-MelB interaction in the presence of cations.     

Substrate-induced conformational changes in Escherichia coli arginyl-tRNA synthetase observed by 19F NMR spectroscopy

The 19F nuclear magnetic resonance (NMR) spectra of 4-fluorotryptophan (4-F-Trp)-labeled Escherichia coli arginyl-tRNA synthetase (ArgRS) show that there are distinct conformational changes in the catalytic core and tRNA anticodon stem and loop-binding domain of the enzyme, when arginine and tRNA(Arg) are added to the unliganded enzyme. We have assigned five fluorine resonances of 4-F-Trp residues (162, 172, 228, 349 and 446) in the spectrum of the fluorinated enzyme by site-directed mutagenesis. The local conformational changes of E. coli ArgRS induced by its substrates observed herein by 19F NMR are similar to those of crystalline yeast homologous enzyme.     

Enhanced conversion rate of L-phenylalanine by coupling reactions of aminotransferases and phosphoenolpyruvate carboxykinase in Escherichia coli K-12.

In Escherichia coli, aspartate aminotransferase (encoded by aspC) and aromatic amino acid aminotransferase (encoded by tyrB) share overlapping substrate specificity in the syntheses of aromatic amino acids. Through the transamination reactions catalyzed by AspC or TyrB, L-phenylalanine (L-Phe) can be produced from phenylpyruvate with aspartic acid as the amino donor. To modulate and enhance the production levels of proteins, both aspC and tyrB were subcloned into a runaway-replication vector. As a result, the specific activities of AspC and TyrB obtained showed 65-fold and 50-fold increases, respectively, compared with the wild-type level. Employing resting cells of AspC- and TyrB-overproducing E. coli K-12 strains for L-Phe productions resulted in molar conversion yields of 70% and 55%, respectively. With an additional introduction of phosphoenolpyruvate carboxykinase (encoded by pck) into the transamination reactions, the conversion yields were improved to 93% from 70% and to 75% from 55% in a relatively short time. These results account for more than an 8-fold increase in productivity, as compared to the previous report (Calton et al., 1985). In addition, a four-run reuse of the recombinant cells for L-Phe production gave a total yield of 91 g/L with a 93% conversion.     

Mechanisms of activation of phosphoenolpyruvate carboxykinase from Escherichia coli by Ca2+ and of desensitization by trypsin

The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.     

Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli.

cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached to Cys178. Binding of cAMP in the CRP-(cAMP)2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP-(cAMP)4 complex alters the surroundings of Trp85. Time-resolved anisotropy measurements indicated that cAMP binding in the CRP-(cAMP)2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP-(cAMP)2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 A in apo-CRP to 18.7 A in the CRP-(cAMP)2 complex. The binding of cAMP in the CRP-(cAMP)4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP-DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to approximately 20 A. The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein.     

Monitoring protein conformational changes by quenching of intrinsic fluorescence

The quenching of intrinsic fluorescence of human serum albumin and pigeon liver malic enzyme by acrylamide was studied after the proteins were denatured to different stages. The progress of protein denaturation induced by guanidine hydrochloride was accompanied by increasing of effective dynamic quenching constant which provides a convenient parameter for monitoring protein conformational change.     

Nucleotide specificity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase Kinetics, fluorescence spectroscopy, and molecular simulation studies

Phosphoenolpyruvate carboxykinases, depending on the enzyme origin, preferentially use adenine or guanine nucleotides as substrates. In this work, analyses of the substrate specificity of the Saccharomyces cerevisiae ATP-dependent enzyme have been carried out. Kinetics studies gave relative values of k(cat)/K(m) for the nucleoside triphosphate complexes in the order ATP>GTP>ITP>UTP>CTP. For the nucleoside diphosphate complexes the order is ADP>GDP>IDP congruent withUDP>CDP. This shows that the enzyme has a strong preference for ADP (or ATP) over other nucleotides, being this preference about an order of magnitude higher for the diphosphorylated than for the triphosphorylated nucleosides. The calculated binding free energies (kcalmol(-1)) at 25 degrees C are 7.39 and 6.51 for ATP and ADP, respectively. These values decrease with the nucleotide structure in the same order than the kinetic specificity. The binding energy for any triphosphorylated nucleoside is more favourable than for the corresponding diphosphorylated compound, showing the relevance of the P(gamma) for nucleotide binding. Homology models of the adenine and guanine nucleotides in complex with the enzyme show that the base adopts a similar conformation in the diphosphorylated nucleosides while in the triphosphorylated nucleosides the sugar-base torsion angle is 61 degrees for ATP and -53 degrees for GTP. Differences are also noted in the distance between P(beta) and Mn2+ at site 1. This distance is almost the same in the ATP, GTP, and UTP complexes, however in the ADP, GDP and UDP complexes it is 2.9, 5.1, and 7A, respectively. Experimental data obtained with a Thr463Ala mutant enzyme agree with molecular simulation predictions. The results here presented are discussed in terms of the proposed interactions of the nucleotides with the protein.     

Detergent-induced conformational changes of Humicola lanuginosa lipase studied by fluorescence spectroscopy

Detergent (pentaoxyethylene octyl ether, C(8)E(5))-induced conformational changes of Humicola lanuginosa lipase (HLL) were investigated by stationary and time-resolved fluorescence intensity and anisotropy measurements. Activation of HLL is characterized by opening of a surface loop (the "lid") residing directly over the enzyme active site. The interaction of HLL with C(8)E(5) increases fluorescence intensities, prolongs fluorescence lifetimes, and decreases the values of steady-state anisotropy, residual anisotropy, and the short rotational correlation time. Based on these data, we propose the following model. Already below critical micellar concentration (CMC) the detergent can intercalate into the active site accommodating cleft, while the lid remains closed. Occupation of the cleft by C(8)E(5) also blocks the entry of the monomeric substrate, and inhibition of catalytic activity at [C(8)E(5)] less than or equal to CMC is evident. At a threshold concentration close to CMC the cooperativity of the hydrophobicity-driven binding of C(8)E(5) to the lipase increases because of an increase in the number of C(8)E(5) molecules present in the premicellar nucleates on the hydrophobic surface of HLL. These aggregates contacting the lipase should have long enough residence times to allow the lid to open completely and expose the hydrophobic cleft. Concomitantly, the cleft becomes filled with C(8)E(5) and the "open" conformation of HLL becomes stable.     

Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold.     

Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

The stereochemistry of the carboxylation of phosphoenolpyruvate to yield oxalacetate, catalyzed by chicken liver phosphoenolpyruvate carboxykinase and by Ascaris muscle phosphoenolpyruvate carboxykinase, was determined. The substrate (Z)-3-fluorophosphoenolpyruvate was used for the stereochemical analysis. The carboxylation reaction was coupled to malate dehydrogenase to yield 3-fluoromalate, and the stereochemistry of the products was identified by 19F NMR. In separate experiments, the enantiomeric tautomers of 3-fluorooxalacetate were shown to be utilized by malate dehydrogenase to yield (2R,3R)- and (2R,3S)-3-fluoromalate in nearly identical amounts. The products were identified by 19F NMR. When (Z)-3-fluorophosphoenolpyruvate was used as a substrate for phosphoenolpyruvate carboxykinase from avian liver and from Ascaris, and malate dehydrogenase was used to trap the product, only a single diastereomer was observed. This product was shown to be (2R,3R)-3-fluoromalate in each case. The assignments were based on coupling constants taken from Keck et al. [Keck, R., Hess, H., & Rétey, J. (1980) FEBS Lett. 114, 287]. These results indicate that the stereochemistry of carboxylation, catalyzed by chicken phosphoenolpyruvate carboxykinase and by Ascaris phosphoenolpyruvate carboxykinase, is identical and takes place from the si side of the enzyme-bound phosphoenolpyruvate. The carboxylation reaction was run both in H2O and in D2O. No deuterium incorporation into fluoromalate was shown to occur. The product 3-fluorooxalacetate is thus released from phosphoenolpyruvate carboxykinase as the keto form and is reduced more rapidly by reduced nicotinamide adenine dinucleotide with malate dehydrogenase than by the occurrence of tautomerization.