Molecular and biochemical characterization of natural and recombinant phosphoglycerate kinase B from Trypanosoma rangeli.

T. rangeli epimastigotes contain only a single detectable phosphoglycerate kinase (PGK) enzyme in their cytosol. Analysis of this parasite's recently sequenced genome showed a gene predicted to code for a PGK with the same molecular mass as the natural enzyme, and with a cytosolic localization as well. In this work, we have partially purified the natural PGK from T. rangeli epimastigotes. Furthermore, we cloned the predicted PGK gene and expressed it as a recombinant active enzyme. Both purified enzymes were kinetically characterized and displayed similar substrate affinities, with KmATP values of 0.13 mM and 0.5 mM, and Km3PGA values of 0.28 mM and 0.71 mM, for the natural and recombinant enzyme, respectively. The optimal pH for activity of both enzymes was in the range of 8-10. Like other PGKs, TrPGK is monomeric with a molecular mass of approximately 44 kDa. The enzyme's kinetic characteristics are comparable with those of cytosolic PGK isoforms from related trypanosomatid species, indicating that, most likely, this enzyme is equivalent with the PGKB that is responsible for generating ATP in the cytosol of other trypanosomatids. This is the first report of a glycolytic enzyme characterization from T. rangeli.

Molecular cloning, purification and characterization of Brugia malayi phosphoglycerate kinase.

Phosphoglycerate kinase (PGK) is a glycolytic enzyme present in many parasites. It has been reported as a candidate molecule for drug and vaccine developments. In the present study, a full-length cDNA encoding the Brugia malayi 3-phosphoglycerate kinase (BmPGK) with an open reading frame of 1.3 kb was isolated and PCR amplified and cloned. The exact size of the BmPGK's ORF is 1377 bps. The BmPGK gene was subcloned into pET-28a (+) expression vector, the expressed enzyme was purified by affinity column and characterized. The SDS-PAGE analysis revealed native molecular weight of recombinant Brugia malayi 3-phosphoglycerate kinase (rBmPGK) to be ∼45 kDa. The enzyme was found sensitive to temperature and pH, it showed maximum activity at 25 °C and pH 8.5. The Km values for PGA and ATP were 1.77 and 0.967 mM, respectively. The PGK inhibitor, clorsulon and antifilarial drugs albendazole and ivermectin inhibited the enzyme. The specific inhibitor of PGK, clorsulon, competitively inhibited enzyme with Ki value 1.88 µM. Albendazole also inhibited PGK competitively with Ki value 35.39 µM. Further these inhibitory studies were confirmed by docking and molecular simulation of drugs with enzyme. Clorsulon interacted with substrate binding site with glutamine 37 as well as in hinge regions with aspartic acid 385 and valine 387 at ADP binding site. On the other hand albendazole interacted with asparagine 335 residues. These effects were in good association with binding interactions. Thus current study might help in designing and synthesis of effective inhibitors for this novel drug target and understanding their mode of interaction with the potent anthelmintic drugs.

Trypanosoma evansi contains two auxiliary enzymes of glycolytic metabolism: Phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase.

Trypanosoma evansi is a monomorphic protist that can infect horses and other animal species of economic importance for man. Like the bloodstream form of the closely related species Trypanosoma brucei, T. evansi depends exclusively on glycolysis for its free-energy generation. In T. evansi as in other kinetoplastid organisms, the enzymes of the major part of the glycolytic pathway are present within organelles called glycosomes, which are authentic but specialized peroxisomes. Since T. evansi does not undergo stage-dependent differentiations, it occurs only as bloodstream forms, it has been assumed that the metabolic pattern of this parasite is identical to that of the bloodstream form of T. brucei. However, we report here the presence of two additional enzymes, phosphoenolpyruvate carboxykinase and PPi-dependent pyruvate phosphate dikinase in T. evansi glycosomes. Their colocalization with glycolytic enzymes within the glycosomes of this parasite has not been reported before. Both enzymes can make use of PEP for contributing to the production of ATP within the organelles. The activity of these enzymes in T. evansi glycosomes drastically changes the model assumed for the oxidation of glucose by this parasite.

Characterization of 3-phosphoglycerate kinase from Corynebacterium glutamicum and its impact on amino acid production.

BACKGROUND: Corynebacterium glutamicum cg1790/pgk encodes an enzyme active as a 3-phosphoglycerate kinase (PGK) (EC 2.7.2.3) catalyzing phosphoryl transfer from 1,3-biphosphoglycerate (bPG) to ADP to yield 3-phosphoglycerate (3-PG) and ATP in substrate chain phosphorylation. RESULTS: C. glutamicum 3-phosphoglycerate kinase was purified to homogeneity from the soluble fraction of recombinant E. coli. PGK(His) was found to be active as a homodimer with molecular weight of 104 kDa. The enzyme preferred conditions of pH 7.0 to 7.4 and required Mg²âº for its activity. PGK(His) is thermo labile and it has shown maximal activity at 50-65°C. The maximal activity of PGK(His) was estimated to be 220 and 150 U mg-1 with KM values of 0.26 and 0.11 mM for 3-phosphoglycerate and ATP, respectively. A 3-phosphoglycerate kinase negative C. glutamicum strain ∆pgk was constructed and shown to lack the ability to grow under glycolytic or gluconeogenic conditions unless PGK was expressed from a plasmid to restore growth. When pgk was overexpressed in L-arginine and L-ornithine production strains the production increased by 8% and by 17.5%, respectively. CONCLUSION: Unlike many bacterial PGKs, C. glutamicum PGK is active as a homodimer. PGK is essential for growth of C. glutamicum with carbon sources requiring glycolysis and gluconeogenesis. Competitive inhibition by ADP reveals the critical role of PGK in gluconeogenesis by energy charge. Pgk overexpression improved the productivity in L-arginine and L-ornithine production strains.

Expression, purification, crystallization and preliminary crystallographic analysis of the phosphoglycerate kinase from Acinetobacter baumannii.

Acinetobacter baumannii is a common multidrug-resistant clinical pathogen that is often found in hospitals. The A. baumannii phosphoglycerate kinase (AbPGK) is involved in the key energy-producing pathway of glycolysis and presents a potential target for antibiotic development. AbPGK has been expressed and purified; it was crystallized using lithium sulfate as the precipitant. The AbPGK crystals belonged to space group P222(1). They diffracted to a resolution of 2.5 Šusing synchrotron radiation at the Canadian Light Source.

Phosphoglycerate kinase and fructose bisphosphate aldolase of Candida albicans as new antigens recognized by human salivary IgA.

BACKGROUND: Candida albicans is an opportunistic dimorphic fungus commonly present in the human oral cavity that causes infections in immunocompromised patients. The antigen variability, influenced by growth conditions, is a pathogenicity factor. AIMS: To determine the effect of nutritional and heat stress on the antigen expression of C. albicans, and to identify major antigens recognized by human salivary secretory immunoglobulin A (sIgA). METHODS: Under various different nutritional conditions, heat shock was induced in C. albicans cells in stationary and exponential growth phases. The expression of protein determinants of C. albicans was assessed by Western blot analysis against human saliva. The antigens were purified and characterized by two-dimensional electrophoresis and identified by protein microsequencing. RESULTS: Five antigens recognized by salivary IgA were characterized as mannoproteins due to their reactivity with concanavalin A. They did not show reactivity with anti-heat shock protein monoclonal antibodies. Two of them (42 and 36 kDa) were found to be regulated by heat shock and by nutritional stress and they were identified as phosphoglycerate kinase and fructose bisphosphate aldolase, respectively. CONCLUSIONS: These glycolytic enzymes are major antigens of C. albicans, and their differential expression and recognition by the mucosal immune response system could be involved in protection against oral infection.

Expression, purification, crystallization and preliminary X-ray diffraction studies of phosphoglycerate kinase from methicillin-resistant Staphylococcus aureus MRSA252.

Phosphoglycerate kinase (PGK) from methicillin-resistant Staphylococcus aureus MRSA252 has been cloned in pQE30 expression vector, overexpressed in Escherichia coli SG13009 (pREP4) cells and purified to homogeneity. The protein was crystallized from 0.15 M CaCl(2), 0.1 M HEPES-NaOH pH 6.8, 20%(w/v) polyethylene glycol 2000 at 298 K by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a = 45.14, b = 74.75, c = 58.67 Å, ß = 95.72°. X-ray diffraction data have been collected and processed to a maximum resolution of 2.3 Å. The presence of one molecule in the asymmetric unit gives a Matthews coefficient (V(M)) of 2.26 Å(3) Da(-1) with a solvent content of 46%. The structure has been solved by molecular replacement and structure refinement is now in progress.

Recovery of functional enzyme from amyloid fibrils.

Amyloid deposits, which accumulate in numerous diseases, are the final stage of multi-step protein conformational-conversion and oligomerization processes. The underlying molecular mechanisms are not fully understood, and particularly little is known about the reverse reaction. Here we show that phosphoglycerate kinase amyloid fibrils can be converted back into native protein. We achieved recovery with 60% efficiency, which is comparable to the success rate of the unfolding-refolding studies, and the recovered enzyme was folded, stable and fully active. The key intermediate stages in the recovery process are fibril disassembly and unfolding followed by spontaneous protein folding.

Chloroplast phosphoglycerate kinase from Euglena gracilis: endosymbiotic gene replacement going against the tide.

Two chloroplast phosphoglycerate kinase isoforms from the photosynthetic flagellate Euglena gracilis were purified to homogeneity, partially sequenced, and subsequently cDNAs encoding phosphoglycerate kinase isoenzymes from both the chloroplast and cytosol of E. gracilis were cloned and sequenced. Chloroplast phosphoglycerate kinase, a monomeric enzyme, was encoded as a polyprotein precursor of at least four mature subunits that were separated by conserved tetrapeptides. In a Neighbor-Net analysis of sequence similarity with homologues from numerous prokaryotes and eukaryotes, cytosolic phosphoglycerate kinase of E. gracilis showed the highest similarity to cytosolic and glycosomal homologues from the Kinetoplastida. The chloroplast isoenzyme of E. gracilis did not show a close relationship to sequences from other photosynthetic organisms but was most closely related to cytosolic homologues from animals and fungi.

Flow process for electroextraction of intracellular enzymes from the fission yeast, Schizosaccharomyces pombe.

Flow treatment of the yeast, Schizosaccharomyces pombe, with high intensity electric field pulses released intracellular enzymes such as glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. Over 70% of the total activity was liberated within 4 h after pulse application. The optimal field intensities were considerably higher than that needed for irreversible plasma membrane permeabilization.

Regulation of urokinase receptor expression by phosphoglycerate kinase.

Post-transcriptional regulation represents a major mechanism by which eukaryotic gene expression is regulated through cis-trans interactions that serve as signals for rapid alterations of messenger RNA (mRNA) stability. Regulation of urokinase-type plasminogen activator receptor (uPAR) mRNA involves the interaction of a uPAR mRNA coding region sequence with a 50 kD uPAR mRNA binding protein. We purified this protein from human bronchial epithelial (Beas2B) cells and identified it as phosphoglycerate kinase (PGK). We cloned PGK cDNA by polymerase chain reaction and expressed the recombinant PGK protein, which specifically bound the uPAR mRNA coding region by gel mobility shift and Northwestern blotting. We also confirmed a direct interaction of PGK protein with uPAR mRNA by immunoprecipitation. Overexpression of PGK in uPAR-overproducing H157 lung carcinoma cells resulted in decreased cytoplasmic uPAR mRNA and cell surface uPAR protein expression. Reduced uPAR mRNA expression involved decreased stability of the uPAR mRNA. Decline in 3H-thymidine incorporation and migration occurred in H157 cells transfected with PGK cDNA. These results demonstrate that PGK regulates uPAR expression at the post-transcriptional level.

Crystallization and preliminary X-ray crystallographic studies of phosphoglycerate kinase from Thermus caldophilus.

We report the purification and crystallization of phosphoglycerate kinase from Thermus caldophilus (Tca). The enzyme crystallizes in the P2(1)2(1)2(1) space group (cell dimensions a = 65.1, b = 71.3, c = 80.2 A), with one molecule in the asymmetric unit. A complete set of diffraction data was collected from an orthorhombic crystal up to 1.8 A resolution.

Nucleotide binding to pig muscle 3-phosphoglycerate kinase in the crystal and in solution: relationship between substrate antagonism and interdomain communication.

Binding constants for the nucleotide substrates were determined in two different crystalline forms of pig muscle 3-phosphoglycerate kinase (PGK): the binary complex with 3-phosphoglycerate (3-PG) in which the two domains are in an open conformation (Harlos, Vas, and Blake (1992) Proteins, 12, 133-144) and the ternary complex with 3-PG and the Mg salt of the ATP analogue, beta,gamma-methyleneadenosine-5'-triphosphate (AMP-PCP), the structure of which is under resolution. Competitive titrations have been performed in the presence of the chromophoric analogue of ATP, 2'3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP), similar to those previously carried out in solution, where a weakening of the binding of the nucleotide substrates in the presence of the other substrate, 3-PG, has been observed (Vas, Merli, and Rossi (1994) Biochem. J. 301, 885-891). Here the K(d) values for MgADP were found to be 0.096 +/- 0.021 and 0.045 +/- 0.016 mM, respectively, for the crystals of the binary and ternary complexes. Both K(d) values are significantly smaller than the one obtained in solution in the presence of 3-PG (0.38 +/- 0.05 mM) and are close to the values determined in solution in the absence of 3-PG (0.06 +/- 0.01 mM). Thus, the "substrate antagonism" observed in solution is not present in either of the investigated crystal forms. Further nucleotide binding studies with the solubilized enzyme have shown that 3-PG has no effect on ADP (Mg(2+)-free) binding (K(d) = 0.34 +/- 0.05 mM), while it weakens MgADP binding. Thus, 3-PG abolishes the strengthening effect of the Mg(2+) ion on the binding of ADP. This phenomenon is apparently due to the interaction between the carboxyl group of 3-PG and the protein, since the carboxyl-lacking analogue glycerol-3-phosphate has no detectable effect on MgADP binding. Comparison of the crystallographic data of different PGK binary (with either 3-PG or MgADP) and ternary (with both 3-PG and MgADP) complexes, having open and closed conformations, respectively, provides a possible structural explanation of the substrate antagonism. We suggest that the specific interaction between the 3-PG carboxylic group and a conserved arginine side chain is changed during domain closure, and, through interdomain communication, this change may be transmitted to the site in which Mg(2+) binds the ADP phosphates. This effect is abolished in the crystals of pig muscle PGK, in which lattice forces stabilize the open domain conformation.

Purification and characterization of 3-phosphoglycerate kinase from Ehrlich ascites carcinoma cells.

3-Phosphoglycerate kinase (3-PGK) has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The enzyme has been partially characterized and compared with the characteristics of this enzyme of other normal and malignant cells. The EAC cell 3-PGK is composed of a single subunit of 47 kDa. It has a broad pH optimum (pH 6.0-7.5) for its enzymatic activity. The apparent Km values of 3-phosphoglycerate (3-PGA) and ATP for 3-PGK have been found out to be 0.25 mM and 0.1 mM respectively. Similar to 3-PGK of other cells, the EAC enzyme requires either Mg2+ or Mn2+ for full activity; the optimum concentrations of Mg2+ and Mn2+ are 0.8 mM and 0.5 mM respectively. When ATP and 3-PGA act as substrates, ADP, the reaction product of 3-PGK-catalyzed reaction has been found to inhibit this enzyme. Kinetic studies were made on the inhibition of ADP in presence of the substrates ATP and 3-PGA. Attempts to hybridize 3-PGK and glyceraldehyde-3-phosphate dehydrogenase of EAC cells by NAD or glutaraldehyde were unsuccessful.

Enolase, a cellular glycolytic enzyme, is required for efficient transcription of Sendai virus genome.

Cellular proteins (host factors) may play key roles in transcription of Sendai virus (SeV) genome. We have previously shown that the host factor activity, which stimulates in vitro mRNA synthesis of SeV, from bovine brain comprises at least three complementary factors, and two of them were identified as tubulin and phosphoglycerate kinase (PGK). Here the third host factor activity was further resolved into two complementary factors, and one of them was purified to an almost single polypeptide chain with an apparent M(r) of 52,000 (p52) and was identified as a glycolytic enzyme, enolase. Recombinant human alpha-enolase, as did p52, acted synergistically with other three host factors to stimulate SeV mRNA synthesis. West-Western blot analysis demonstrated that tubulin specifically binds enolase as well as PGK, suggesting that these two glycolytic enzymes regulate SeV transcription through their interactions with tubulin.

Involvement of a cellular glycolytic enzyme, phosphoglycerate kinase, in Sendai virus transcription.

In vitro mRNA synthesis of Sendai virus is almost entirely dependent on the addition of cellular proteins (host factors). Previous studies indicated that the host factor activity from bovine brain was resolved into at least two complementary fractions, one of which may be tubulin. In this study, the host factor activity that stimulates the transcription in the presence of tubulin was further purified from bovine brain. This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparent M(r) of 46,000 (p46). From the amino acid sequence, biochemical, and immunological analyses, p46 was identified as a glycolytic enzyme, phosphoglycerate kinase (PGK). Purified native PGK from rabbit and yeast, and a recombinant human PGK substituted for p46. Although, as previously suggested, tubulin was involved in the transcription initiation complex formation by being integrated into the complex, p46 and its complementary factor had little effect on the complex formation. On the other hand, when p46 and the complementary factor were added to the RNA chain elongation reaction from the isolated initiation complex formed with tubulin, mRNA synthesis was dramatically stimulated. The enzymatic activity per se of PGK did not seem to be required for its activity. West-Western blot analysis showed that PGK could directly interact with tubulin. These data suggest that PGK stimulates Sendai virus mRNA synthesis at the elongation step, probably through its interaction with tubulin in the initiation complex.

Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima: catalytic, spectral and thermodynamic properties.

Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima (TmPGK) has been expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity applying heat incubation of the crude extract at 80 degreesC, ion exchange chromatography and gel filtration. The biochemical, catalytic and spectral properties were compared with those of the natural enzyme and found to be identical. As shown by SDS-PAGE, ultracentrifugal analysis and gel filtration chromatography, the enzyme is a 43 kDa monomer. At neutral pH, the guanidinium chloride (GdmCl) and temperature-induced denaturation transitions reveal two-state behaviour with high cooperativity. As taken from the temperature dependence of the free energy of unfolding at zero GdmCl concentration and pH 7, optimum stability is observed at approximately 30 degreesC. The difference in the free energies of stabilization for the enzymes from yeast and Thermotoga amounts to Delta DeltaG=85 kJ/mol. The extrapolated temperatures of cold and heat-denaturation are about -10 and +85 degreesC. This indicates that the stability profile of TmPGK is shifted to higher free energy values and broadened over a wider temperature range, compared to that observed for PGKs from mesophiles or moderately thermophiles. In order to achieve cold or heat-denaturation, GdmCl concentrations of approximately 1.8 or approximately 0.9 M are required. Due to a kinetic intermediate on the pathway of cold denaturation, equilibration in the transition range takes exceedingly long.