Formation Of Phosphoenolpyruvate Research Articles

Molecular and biochemical characterization of the sunflower (Helianthus annuus L.) cytosolic and plastidial enolases in relation to seed development

In the present study, we describe the molecular and biochemical characterization of sunflower (Helianthus annuus L.) enolase (ENO, EC 4.2.1.11) proteins, which catalyze the formation of phosphoenolpyruvate, the penultimate intermediate in the glycolytic pathway. We cloned and characterized three cDNAs encoding different ENO isoforms from developing sunflower seeds. Studies using fluorescently tagged ENOs confirmed the predicted subcellular localization of ENO isoforms: HaENO1 in the plastid while HaENO2 and HaENO3 were found in the cytosol. The cDNAs were used to express the corresponding 6(His)-tagged proteins in Escherichia coli. The proteins were purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Recombinant HaENO1 and HaENO2, but not HaENO3 were shown to have enolase activity, in agreement with data obtained with the Arabidopsis homolog proteins. Site directed mutagenesis of several critical amino acids was used to attempt to recover enolase activity in recombinant HaENO3, resulting in very small increases that were not additive. A kinetic characterization of the two active isoforms showed that pH had similar effect on their velocity, that they had similar affinity for 2-phosphoglycerate, but that the kcat/Km of the plastidial enzyme was higher than that of the cytosolic isoform. Even though HaENO2 was always the most highly expressed transcript, the levels of expression of the three ENO genes were remarkably distinct in all the vegetative and reproductive tissues studied. This indicates that in seeds the conversion of 2-phosphoglycerate to phosphoenolpyruvate takes place through the cytosolic and the plastidial pathways therefore both routes could contribute to the supply of carbon for lipid synthesis. The identity of the main source of carbon during the period of stored products synthesis is discussed.

Trapped intermediate state of plant pyruvate phosphate dikinase indicates substeps in catalytic swiveling domain mechanism.

Pyruvate phosphate dikinase (PPDK) is an essential enzyme of both the C4 photosynthetic pathway and cellular energy metabolism of some bacteria and unicellular protists. In C4 plants, it catalyzes the ATP- and Pi -dependent formation of phosphoenolpyruvate (PEP) while in bacteria and protozoa the ATP-forming direction is used. PPDK is composed out of three distinct domains and exhibits one of the largest single domain movements known today during its catalytic cycle. However, little information about potential intermediate steps of this movement was available. A recent study resolved a discrete intermediate step of PPDK's swiveling movement, shedding light on the details of this intriguing mechanism. Here we present an additional structural intermediate that possibly represents another crucial step in the catalytic cycle of PPDK, providing means to get a more detailed understanding of PPDK's mode of function.

Structural intermediates and directionality of the swiveling motion of Pyruvate Phosphate Dikinase

Pyruvate phosphate dikinase (PPDK) is a vital enzyme in cellular energy metabolism catalyzing the ATP- and P i -dependent formation of phosphoenolpyruvate from pyruvate in C4 -plants, but the reverse reaction forming ATP in bacteria and protozoa. The multi-domain enzyme is considered an efficient molecular machine that performs one of the largest single domain movements in proteins. However, a comprehensive understanding of the proposed swiveling domain motion has been limited by not knowing structural intermediates or molecular dynamics of the catalytic process. Here, we present crystal structures of PPDKs from Flaveria, a model genus for studying the evolution of C4 -enzymes from phylogenetic ancestors. These structures resolve yet unknown conformational intermediates and provide the first detailed view on the large conformational transitions of the protein in the catalytic cycle. Independently performed unrestrained MD simulations and configurational free energy calculations also identified these intermediates. In all, our experimental and computational data reveal strict coupling of the CD swiveling motion to the conformational state of the NBD. Moreover, structural asymmetries and nucleotide binding states in the PPDK dimer support an alternate binding change mechanism for this intriguing bioenergetic enzyme.

Description of carbon fixation pathway based on <italic>Skeletonema marinoi</italic> transcriptome

Diatoms can fix approximately 1016 g CO2 into organic carbon every year, equivalent to roughly 20% of global primary production. They are the basis for important food webs, having considerable impacts on carbon cycle and biological carbon pump that draws down CO2 from the atmosphere to the ocean interior. Nevertheless, the K 1/2 of their ribulose- 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is 40~60 μ mol L - 1, higher than the apparent photosynthetic K 1/2 (CO2) for ambient CO2. Growth and kinetic studies have shown that diatoms can avoid CO2 limitation through CO2 concentrating mechanisms. Recently the carbon fixation metabolism pathway in diatoms is further elucidated by the presence of a complete set of genes essential for this metabolic route in Thalassiosira pseudonana and Phaeodactylum tricornutum because of their entire availablegenome information. In this study, we used RNA-Seq technology to confirm the existence of necessary genes for plant-like C4 activity in Skeletonema marinoi , which is a complex biological trait that enables S. marinoi to either has high water-use efficiency or accumulate biomass at a fast rate. The carbon fixation metabolism pathway of S. marinoi involves 20 enzymes and 34 coding genes, including the complete key enzymes especially for C4 pathway, which are phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate orthophosphate dikinase (PPDK). Comparing with the homologous genes of carbon fixation metabolism pathway in T. pseudonana and P. tricornutum , these genes showed little divergence. Besides, the gene differential expression of S. marinoi in carbon fixation metabolism pathway was identified at growth stages using digital gene expression profiling. The results indicated the number of differential expression genes was 7 and 3 in C3 and C4 pathway, respectively. As for the differential expression of coding genes in growth stages, there were 8 and 10 coding genes in the stationary and decline phase, respectively versus to those in the exponential phase. The gene expression of fructose-1,6-bisphosphatase (FBP), fructose-bisphosphate aldolase (ALDO) and PPDK was changed significantly after the exponential phase. The FBP and ALDO are not only responsible for the carbon fixation metabolism pathway, but also as the key enzymes in the glycolytic metabolism pathway, which links closely with the flow direction of carbon and the synthesis of carbohydrates. The PPDK, a cardinal enzyme of the C4 pathway, catalyzing the regeneration of phosphoenol pyruvate(PEP), evolved as an adaptation to high light intensity, high temperature, low concentration of CO2 and dryness. Therefore, the formation of PEP through PPDK is considered to be very important in the C4 pathway. This study will contribute to analyze the gene regulation of key enzymes involved in the carbon metabolism of S. marinoi , explain the reason for huge amounts of primary production by diatom, and enhance our knowledge on the carbon fixation mechanism, which provides a new insight into understanding carbon biogeochemical cycle.

Physiological efficiency of succulents under different growing environments in tropical condition

Investigations were undertaken to study the physiological effectiveness of different ornamental succulents under tropical condition. Six species of succulents were evaluated under four different growing environments. Results showed that the performance of succulents grown under conservatory house condition was found to be performed well and ideal with respect to chlorophyll, carotenoids, relative water content and photosynthetic rate than other growing environments. The succulent species viz ., Haworthia limifolia, Monadenium lugardiae and Aloe juvenna were the most suitable species for growing under conservatory house condition due to its high nocturnal CO 2 uptake and cell sap acidification which possess CAM metabolism. CAM involves nocturnal CO 2 uptake and fixation by cytosolic phosphoenolpyruvate carboxylase into malic acid that is stored in the vacuole causing nocturnal cell sap acidification. During the subsequent light period, malic acid is released from the vacuole in the form of malate that is then decarboxylated leading to deacidification of cell sap, formation of pyruvate or phosphoenolpyruvate and liberation of CO 2 . Repeated acidification and deacidification cycles represent a characteristic physiological feature of CAM metabolism in succulents which is best suited for xeric environment and possess high photosynthetic efficiency.

Model-guided metabolic gene knockout of gnd for enhanced succinate production in Escherichia coli from glucose and glycerol substrates

The metabolic role of 6-phosphogluconate dehydrogenase (gnd) under anaerobic conditions with respect to succinate production in Escherichia coli remained largely unspecified. Herein we report what are to our knowledge the first metabolic gene knockout of gnd to have increased succinic acid production using both glucose and glycerol substrates in E. coli. Guided by a genome scale metabolic model, we engineered the E. coli host metabolism to enhance anaerobic production of succinic acid by deleting the gnd gene, considering its location in the boundary of oxidative and non-oxidative pentose phosphate pathway. This strategy induced either the activation of malic enzyme, causing up-regulation of phosphoenolpyruvate carboxylase (ppc) and down regulation of phosphoenolpyruvate carboxykinase (ppck) and/or prevents the decarboxylation of 6 phosphogluconate to increase the pool of glyceraldehyde-3-phosphate (GAP) that is required for the formation of phosphoenolpyruvate (PEP). This approach produced a mutant strain BMS2 with succinic acid production titers of 0.35gl−1 and 1.40gl−1 from glucose and glycerol substrates respectively. This work further clearly elucidates and informs other studies that the gnd gene, is a novel deletion target for increasing succinate production in E. coli under anaerobic condition using glucose and glycerol carbon sources. The knowledge gained in this study would help in E. coli and other microbial strains development for increasing succinate production and/or other industrial chemicals.

Unusual Properties and Functions of Plant Pyruvate, Orthophospate Dikinase

The pyruvate, orthophosphate dikinase (PPDK, EC 2.7.9.1), crucial plant enzyme in the process of concentrating CO2 for Calvin cycle, catalyzes phosphoenolpyruvate-regeneration phase of the C4 photosynthetic pathway. However plants possess also nonphotosynthetic isoform of PPDK, which functions are less understood, probably due to its low abundance. In all plants, PPDK is located in both cytoplasmic and plastidic compartments. PPDK catalyzes the reversible ATP- and Pi- dependent formation of phosphoenolpyruvate from pyruvate.

Structural and functional studies of phosphoenolpyruvate carboxykinase from Mycobacterium tuberculosis.

Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.

The mechanism of oxythiamine-induced collagen biosynthesis in cultured fibroblasts.

The oxythiamine (OXY) is antivitamin of thiamine. The finding that OXY increases the cytoplasmic concentration of pyruvate, known to enhance collagen biosynthesis, led us to investigate the mechanism of this antivitamin action on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with micromolar concentrations (30–1,000 µM) of OXY for 24 and 48 h. It was found that OXY-dependent increase in collagen biosynthesis was accompanied by parallel increase in prolidase activity and level, compared to untreated cells. Since phosphoenolpyruvate (PEP) is known as an inhibitor of prolidase—the enzyme that plays important role in collagen biosynthesis, the mechanism of pyruvate interconversion was considered as a regulatory switch in collagen biosynthesis. In fact, 3-MPA, specific inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), contributed to up-regulation of prolidase activity, suggesting that down-regulation of PEP formation is an underlying mechanism. Since collagen biosynthesis and prolidase activity are regulated by signal induced by activated α2β1 integrin receptor as well as insulin-like growth factor-I receptor (IGF-IR), the expression of these receptors was measured by Western immunoblot analysis. The exposure of the cells to OXY contributed to decrease in IGF-IR, α2β1 integrin receptor, pERK1/2, and NF-κB p65 expressions. It was accompanied by increase in total ERK1/2 expression and induction of phosphorylation of Akt protein. The data suggest that OXY-dependent increase of collagen biosynthesis in cultured human skin fibroblasts results from activation of prolidase activity and level, induction in pAkt expression and down-regulation of pERK1/2 and NF-κB p65, the known inhibitor of collagen gene expression.

On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer

Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3(-) complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3 on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.

Robust Control of PEP Formation Rate in the Carbon Fixation Pathway of C4 Plants by a Bi-functional Enzyme

BackgroundC4 plants such as corn and sugarcane assimilate atmospheric CO2 into biomass by means of the C4 carbon fixation pathway. We asked how PEP formation rate, a key step in the carbon fixation pathway, might work at a precise rate, regulated by light, despite fluctuations in substrate and enzyme levels constituting and regulating this process.ResultsWe present a putative mechanism for robustness in C4 carbon fixation, involving a key enzyme in the pathway, pyruvate orthophosphate dikinase (PPDK), which is regulated by a bifunctional enzyme, Regulatory Protein (RP). The robust mechanism is based on avidity of the bifunctional enzyme RP to its multimeric substrate PPDK, and on a product-inhibition feedback loop that couples the system output to the activity of the bifunctional regulator. The model provides an explanation for several unusual biochemical characteristics of the system and predicts that the system's output, phosphoenolpyruvate (PEP) formation rate, is insensitive to fluctuations in enzyme levels (PPDK and RP), substrate levels (ATP and pyruvate) and the catalytic rate of PPDK, while remaining sensitive to the system's input (light levels).ConclusionsThe presented PPDK mechanism is a new way to achieve robustness using product inhibition as a feedback loop on a bifunctional regulatory enzyme. This mechanism exhibits robustness to protein and metabolite levels as well as to catalytic rate changes. At the same time, the output of the system remains tuned to input levels.

Myc Promoter-Binding Protein-1 (MBP-1) Is a Novel Potential Prognostic Marker in Invasive Ductal Breast Carcinoma

BackgroundAlpha-enolase is a glycolytic enzyme that catalyses the formation of phosphoenolpyruvate in the cell cytoplasm. α-Enolase and the predominantly nuclear Myc promoter-binding protein-1 (MBP-1) originate from a single gene through the alternative use of translational starting sites. MBP-1 binds to the P2 c-myc promoter and competes with TATA-box binding protein (TBP) to suppress gene transcription. Although several studies have shown an antiproliferative effect of MBP-1 overexpression on several human cancer cells, to date detailed observations of α-enolase and MBP-1 relative expression in primary tumors versus normal tissues and their correlation with clinicopathological features have not been undertaken.Methodology and FindingsWe analyzed α-enolase and MBP-1 expression in normal breast epithelium and primary invasive ductal breast carcinoma (IDC) from 177 patients by Western blot and immunohistochemical analyses, using highly specific anti-α-enolase monoclonal antibodies. A significant increase in the expression of cytoplasmic α-enolase was observed in 98% of the tumors analysed, compared to normal tissues. Nuclear MBP-1 was found in almost all the normal tissues while its expression was retained in only 35% of the tumors. Statistically significant associations were observed among the nuclear expression of MBP-1 and ErbB2 status, Ki-67 expression, node status and tumor grade. Furthermore MBP-1 expression was associated with good survival of patients with IDC.ConclusionsMBP-1 functions in repressing c-myc gene expression and the results presented indicate that the loss of nuclear MBP-1 expression in a large number of IDC may be a critical step in the development and progression of breast cancer and a predictor of adverse outcome. Nuclear MBP-1 appears to be a novel and valuable histochemical marker with potential prognostic value in breast cancer.

Metabolic fluxes in the central carbon metabolism of Dinoroseobacter shibae and Phaeobacter gallaeciensis, two members of the marine Roseobacter clade

BackgroundIn the present work the central carbon metabolism of Dinoroseobacter shibae and Phaeobacter gallaeciensis was studied at the level of metabolic fluxes. These two strains belong to the marine Roseobacter clade, a dominant bacterial group in various marine habitats, and represent surface-associated, biofilm-forming growth (P. gallaeciensis) and symbiotic growth with eukaryotic algae (D. shibae). Based on information from recently sequenced genomes, a rich repertoire of pathways has been identified in the carbon core metabolism of these organisms, but little is known about the actual contribution of the various reactions in vivo.ResultsUsing 13C labelling techniques in specifically designed experiments, it could be shown that glucose-grown cells of D. shibae catabolise the carbon source exclusively via the Entner-Doudoroff pathway, whereas alternative routes of glycolysis and the pentose phosphate pathway are obviously utilised for anabolic purposes only. Enzyme assays confirmed this flux pattern and link the lack of glycolytic flux to the absence of phosphofructokinase activity. The previously suggested formation of phosphoenolpyruvate from pyruvate during mixotrophic CO2 assimilation was found to be inactive under the conditions studied. Moreover, it could be shown that pyruvate carboxylase is involved in CO2 assimilation and that the cyclic respiratory mode of the TCA cycle is utilised. Interestingly, the use of intracellular pathways was highly similar for P. gallaeciensis.ConclusionThe present study reveals the first insight into pathway utilisation within the Roseobacter group. Fluxes through major intracellular pathways of the central carbon metabolism, which are closely linked to the various important traits found for the Roseobacter clade, could be determined. The close similarity of fluxes between the two physiologically rather different species might provide the first indication of more general key properties among members of the Roseobacter clade which may explain their enormous success in the marine realm.

Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae alpha-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN(K252 + 255A), SEN(K304A), SEN(K334A), SEN(K344E), SEN(K435L), and SEN(Delta434-435)) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN(K344E), as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN(K344E) had lost alpha-enolase activity, which was also reduced in SEN(K334A) and SEN(Delta434-435). Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN(K252 + 255A), SEN(K435L), and SEN(Delta434-435). The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.

Roles of Asp75, Asp78, and Glu83 of GTP-dependent Phosphoenolpyruvate Carboxykinase from Mycobacterium smegmatis

The roles of Asp(75), Asp(78), and Glu(83) of the (75)DPSDVARVE(83) element of Mycobacterium smegmatis GTP-dependent phosphoenolpyruvate (PEP) carboxykinase (GTP-PEPCK) were investigated. Asp(78) and Glu(83) are fully conserved in GTP-PEP-CKs. The human PEPCK crystal structure suggests that Asp(78) influences Tyr(220); Tyr(220) helps to position bound PEP, and Glu(83) interacts with Arg(81). Experimental data on other PEPCKs indicate that Arg(81) binds PEP, and the phosphate of PEP interacts with Mn(2+) of metal site 1 for catalysis. We found that D78A and E83A replacements severely reduced activity. E83A substitution raised the apparent K(m) value for Mn(2+) 170-fold. In contrast, Asp(75) is highly but not fully conserved; natural substitutions are Ala, Asn, Gln, or Ser. Such substitutions, when engineered, in M. smegmatis enzyme caused the following. 1) For oxaloacetate synthesis, V(max) decreased 1.4-4-fold. K(m) values for PEP and Mn(2+) increased 3-9- and 1.2-10-fold, respectively. K(m) values for GDP and bicarbonate changed little. 2) For PEP formation, V(max) increased 1.5-2.7-fold. K(m) values for oxaloacetate increased 2-2.8-fold. The substitutions did not change the secondary structure of protein significantly. The kinetic effects are rationalized as follows. In E83A the loss of Glu(83)-Arg(81) interaction affected Arg(81)-PEP association. D78A change altered the Tyr(220)-PEP interaction. These events perturbed PEP-Mn(2+) interaction and consequently affected catalysis severely. In contrast, substitutions at Asp(75), a site far from bound PEP, brought subtle effects, lowering oxaloacetate formation rate but enhancing PEP formation rate. It is likely that Asp(75) substitutions affected PEP-Mn(2+) interaction by changing the positions of Asp(78), Arg(81), and Glu(83), which translated to differential effects on two directions.

First characterization of an archaeal GTP-dependent phosphoenolpyruvate carboxykinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.

Phosphoenolpyruvate carboxykinase (PCK), which catalyzes the nucleotide-dependent, reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2, is one of the important enzymes in the interconversion between C3 and C4 metabolites. This study focused on the first characterization of the enzymatic properties and expression profile of an archaeal PCK from the hyperthermophilic archaeon Thermococcus kodakaraensis (PckTk). PckTk showed 30 to 35% identities to GTP-dependent PCKs from mammals and bacteria but was located in a branch distinct from that of the classical enzymes in the phylogenetic tree, together with other archaeal homologs from Pyrococcus and Sulfolobus spp. Several catalytically important regions and residues, found in all known PCKs irrespective of their nucleotide specificities, were conserved in PckTk. However, the predicted GTP-binding region was unique compared to those in other GTP-dependent PCKs. The recombinant PckTk actually exhibited GTP-dependent activity and was suggested to possess dual cation-binding sites specific for Mn2+ and Mg2+. The enzyme preferred phosphoenolpyruvate formation from oxaloacetate, since the Km value for oxaloacetate was much lower than that for phosphoenolpyruvate. The transcription and activity levels in T. kodakaraensis were higher under gluconeogenic conditions than under glycolytic conditions. These results agreed with the role of PckTk in providing phosphoenolpyruvate from oxaloacetate as the first step of gluconeogenesis in this hyperthermophilic archaeon. Additionally, under gluconeogenic conditions, we observed higher expression levels of PckTk on pyruvate than on amino acids, implying that it plays an additional role in the recycling of excess phosphoenolpyruvate produced from pyruvate, replacing the function of the anaplerotic phosphoenolpyruvate carboxylase that is missing from this archaeon.

Cloning and expression of the enolase gene from Desulfovibrio vulgaris (Miyazaki F)

The gene encoding an enolase from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 2.1-kb DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with PstI and BamHI, contained an enolase gene ( eno) and part of the methylenetetrahydrofolate dehydrogenase gene ( folD). The nucleotide sequence of eno indicates that the protein monomer is composed of 434 amino acids. An expression system for eno under control of the T7 promoter was constructed in E. coli. The purified His-tagged enolase formed a homooctamer and was active in the formation of phosphoenolpyruvate (PEP) as well as in the reverse reaction, the formation of d-(+)-2-phosphoglyceric acid (2-PGA). The pH dependence and kinetic properties of the recombinant enolase from the sulfate-reducing bacterium were also studied. The amounts of eno mRNA when the bacterium was grown on glycerol or glucose were compared to that when D. vulgaris was grown on lactate.

Cloning and characterization of bovine cytosolic and mitochondrial PEPCK during transition to lactation.

The cytosolic (C) and mitochondrial (M) forms of phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) are encoded by two different nuclear genes in mouse, human, and chicken. Our objective was to clone the two forms of PEPCK for bovine and determine their expression during the immediate periparturient interval in dairy cows. Bovine PEPCK-C cDNA contains 2,592 nucleotides and contains 84% similarity to the coding sequence of human PEPCK-C cDNA. A 449-nt partial clone of the 3' end of PEPCK-M is 76% similar to the corresponding sequence of human PEPCK-M. The coding sequence of bovine PEPCK-C and coding sequence of the partial PEPCK-M clone were 58% similar but the similarities in the 3'-untranslated regions were negligible. Northern blot analysis revealed single transcripts of 2.85 and 2.35 kb for PEPCK-C and PEPCK-M, respectively. The transition to lactation did not alter PEPCK-M transcript expression, but expression of PEPCK-C mRNA was transiently increased during early lactation, indicating that enhanced hepatic gluconeogenesis during this period may be tied to enhanced capacity for cytosolic rather than mitochondrial formation of phosphoenolpyruvate.

Urea-induced unfolding studies of free- and ligand-bound tetrameric ATP-dependent Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase : Influence of quaternary structure on protein conformational stability

ATP-dependent phosphoenolpyruvate (PEP) carboxykinases are found in plants and microorganisms, and catalyse the reversible formation of PEP, ADP, and CO 2 from oxaloacetate plus ATP. These enzymes vary in quaternary structure although there is significant sequence identity among the proteins isolated from different sources. To help understand the influence of quaternary structure in protein stability, the urea-induced unfolding of free- and substrate-bound tetrameric Saccharomyces cerevisiae PEP carboxykinase is described and compared with the unfolding characteristics of the monomeric Escherichia coli enzyme [Eur. J. Biochem. 255 (1998) 439]. The urea-induced denaturation of S. cerevisiae PEP carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism (CD) spectra, and 1-anilino-8-naphthalenesulfonate (ANS) binding. The unfolding profiles were multi-steps, and formation of hydrophobic structures were detected. The data indicate that unfolding and dissociation of the enzyme tetramer are simultaneous events. Ligand binding, most notably PEP in the presence of MnCl 2, conferred a marked protection against urea-induced denaturation. A similar protection effect was found when N-iodoacetyl- N′-(5-sulfo-1-napthyl)ethylene diamine (1,5-I-AEDANS) was covalently bound at Cys 365, within the active site region. Refolding experiments indicated that total recovery of tertiary structure was only obtained from samples previously unfolded to less than 30%. In the presence of substrates, complete refolding was achieved from samples originally denatured up to 50%. The unfolding behaviour of S. cerevisiae PEP carboxykinase was found to be similar to that of E. coli PEP carboxykinase, however all steps take place at lower urea concentrations. These findings show that, at least for monomeric and tetrameric ATP-dependent PEP carboxykinases, quaternary structure does not contribute to protein conformational stability.

A GTP-dependent Vertebrate-type Phosphoenolpyruvate Carboxykinase from Mycobacterium smegmatis

This is the first report on a bacterial verterbrate-type GTP-dependent phosphoenolpyruvate carboxykinase (PCK). The pck gene of Mycobacterium smegmatis was cloned. The recombinant PCK was overexpressed in Escherichia coli in a soluble form and with high activity. The purified enzyme was found to be monomeric (72 kDa), thermophilic (optimum temperature, 70 degrees C), very stable upon storage at 4 degrees C, stimulated by thiol-containing reducing agents, and inhibited by oxalate and by alpha-ketoglutarate. The requirement for a divalent cation for activity was fulfilled best by Mn(2+) and Co(2+) and poorly by Mg(2+). At 37 degrees C, the highest V(m) value (32.5 units/mg) was recorded with Mn(2+) and in the presence of 37 mm dithiothreitol (DTT). The presence of Mg(2+) (2 mm) greatly lowered the apparent K(m) values for Mn(2+) (by 144-fold in the presence of DTT and by 9.4-fold in the absence of DTT) and Co(2+) (by 230-fold). In the absence of DTT but in the presence of Mg(2+) (2 mm) as the co-divalent cation, Co(2+) was 21-fold more efficient than Mn(2+). For producing oxaloacetate, the enzyme utilized both GDP and IDP; ADP served very poorly. The apparent K(m) values for phosphoenolpyruvate, GDP, and bicarbonate were >100, 66, and 8300 micrometer, respectively, whereas those for GTP and oxaloacetate (for the phosphoenolpyruvate formation activity) were 13 and 12 microm, respectively. Thus, this enzyme preferred the gluconeogenesis/glycerogenesis direction. This property fits the suggestion that in M. smegmatis, pyruvate carboxylase is not anaplerotic but rather gluconeogenic (Mukhopadhyay, B., and Purwantini, E. (2000) Biochim. Biophys. Acta. 1475, 191-206). Both in primary structure and kinetic properties, the mycobacterial PCK was very similar to its vertebrate-liver counterparts and thus could serve as a model for these enzymes; examples for several immediate targets are presented.