Sequencing, high-level expression and phylogeny of the pyrophosphate-dependent phosphofructokinase from the thermophilic spirochete Spirochaeta thermophila

The full-length gene encoding a 554-amino-acid, active pyrophosphate-dependent phosphofructokinase from Spirochaeta thermophila was cloned and sequenced using a combination of degenerate and inverse PCR, and the enzyme expressed to a high level in Escherichia coli. The recombinant enzyme, with a calculated molecular mass of 61 kDa, was purified to near homogeneity and found to be similar to the purified native enzyme for most properties examined. Phylogenetic analysis demonstrated a close relationship between the thermophilic S. thermophila phosphofructokinase and the large beta-subunits of the phosphofructokinases from Borrelia burgdorferi and Treponema pallidum.     

Pyrophosphate-dependent 6-phosphofructokinase, a new glycolytic enzyme in pineapple leaves.

Pineapple leaves contain a pyrophosphate-dependent 6-phosphofructokinase which has been partially purified and characterized. In crude extracts the pyrophosphate-dependent activity is 10 to 20-fold higher than the ATP-dependent activity. The partially purified activity is near 2.5 μmol Fru-1,6-P₂ formed/min/mg protein. In the reaction 1 Fru-1,6-P₂ is formed per 1 pyrophosphate consumed. The enzyme exhibits a pH optimum of 8.0 and the activity is stimulated by Mg⁺⁺. The discovery of a pyrophosphate-dependent 6-phosphofructokinase in pineapple leaves indicates pyrophosphate can serve as an energy source for synthetic reactions in pineapple and perhaps in other plants as well.     

Pyruvate-phosphate dikinase of oxymonads and parabasalia and the evolution of pyrophosphate-dependent glycolysis in anaerobic eukaryotes

In pyrophosphate-dependent glycolysis, the ATP/ADP-dependent enzymes phosphofructokinase (PFK) and pyruvate kinase are replaced by the pyrophosphate-dependent PFK and pyruvate phosphate dikinase (PPDK), respectively. This variant of glycolysis is widespread among bacteria, but it also occurs in a few parasitic anaerobic eukaryotes such as Giardia and Entamoeba spp. We sequenced two genes for PPDK from the amitochondriate oxymonad Streblomastix strix and found evidence for PPDK in Trichomonas vaginalis and other parabasalia, where this enzyme was thought to be absent. The Streblomastix and Giardia genes may be related to one another, but those of Entamoeba and perhaps Trichomonas are distinct and more closely related to bacterial homologues. These findings suggest that pyrophosphate-dependent glycolysis is more widespread in eukaryotes than previously thought, enzymes from the pathway coexists with ATP-dependent more often than previously thought and may be spread by lateral transfer of genes for pyrophosphate-dependent enzymes from bacteria.     

Phosphofructokinase activities within the order Spirochaetales and the characterisation of the pyrophosphate-dependent phosphofructokinase from Spirochaeta thermophila

The subtype of phosphofructokinase activity, either ATP-, ADP- or pyrophosphate-dependent, present in members of three genera from the Spirochaetales was investigated. The individual species/strains examined included Spirochaeta alkalica, S. asiatica, S. halophila, S. isovalerica, S. litoralis, S. zuelzerae, S. thermophila, two thermophilic spirochetes, Treponema bryantii, T. denticola, paragraph signT. pectinovorum, Leptospira biflexa and L. interrogans. All of the Spirochaeta strains, regardless of their phenotype, possessed primarily a pyrophosphate-dependent phosphofructokinase. In contrast, T. bryantii, T. denticola and L. biflexa had predominantly an ATP-dependent activity, whereas no activity was detected in T. pectinovorum or paragraph signL. interrogans. The results suggest that pyrophosphate-dependent phosphofructokinase activity may be a reliable phenotypic marker for the genus Spirochaeta and that there are potentially interesting differences in how the catabolism of saccharides is controlled among members of genera within the Spirochaetales. The pyrophosphate-dependent phosphofructokinase from S. thermophila strain RI 19.B1 was purified (303-fold) to homogeneity and biochemically characterised. The S. thermophila enzyme displayed hyperbolic kinetics with respect to both the forward and reverse cosubstrates and was not significantly affected by traditional activators or inhibitors of phosphofructokinase. The biochemical characterisation represents the first spirochete phosphofructokinase to be described.     

Inhibition of phosphofructokinases by copper(II)

The biochemical inhibition by Cu2+ on eight phylogenetically and biochemically different phosphofructokinases (PFKs) was investigated. The enzymes screened included representatives from thermophilic and mesophilic bacteria, a hyperthermophilic archaeon and a eukaryote, covering all three phosphoryl donor subtypes (ATP, ADP and pyrophosphate). The sensitivities of the enzymes to Cu2+ varied greatly, with the archaeal ADP-PFK being the least and the eukaryote ATP-PFK being the most sensitive. The bacterial ATP- and pyrophosphate-dependent PFKs showed intermediate sensitivity with the exception of the Spirochaeta thermophila enzyme (pyrophosphate-dependent) which was relatively resistant.     

The biochemical properties and phylogenies of phosphofructokinases from extremophiles

The enzyme phosphofructokinase (PFK) is a defining activity of the highly conserved glycolytic pathway, and is present in the domains Bacteria, Eukarya, and Archaea. PFK subtypes are now known that utilize either ATP, ADP, or pyrophosphate as the primary phosphoryl donor and share the ability to catalyze the transfer of phosphate to the 1-position of fructose-6-phosphate. Because of the crucial position in the glycolytic pathway of PFKs, their biochemical characteristics and phylogenies may play a significant role in elucidating the origins of glycolysis and, indeed, of metabolism itself. Despite the shared ability to phosphorylate fructose-6-phosphate, PFKs that have been characterized to date now fall into three sequence families: the PFKA family, consisting of the well-known higher eukaryotic ATP-dependent PFKs together with their ATP- and pyrophosphate-dependent bacterial cousins (including the crenarchaeal pyrophosphate-dependent PFK of Thermoprotetus tenax) and plant pyrophosphate-dependent phosphofructokinases; the PFKB family, exemplified by the minor ATP-dependent PFK activity of Escherichia coli (PFK 2), but which also includes at least one crenarchaeal enzyme in Aeropyrum pernix; and the tentatively named PFKC family, which contains the unique ADP-dependent PFKs from the euryarchaeal genera of Pyrococcus and Thermococcus, which are indicated by sequence analysis to be present also in the methanogenic species Methanococcus jannaschii and Methanosarcina mazei.     

Fructose 6-phosphate metabolism in plants

The kinetic and regulatory properties of the ATP-dependent phosphofructokinase from various plant tissues are reviewed. Particular attention is given to the differences in properties between the plastid and cytosolic isozymes of this enzyme. A model for fructose 6-phosphate utilization in plants is presented which incorporates a role for the pyrophosphate-dependent phosphofructokinase.     

Purification, microsequencing and cloning of spinach ATP-dependent phosphofructokinase link sequence and function for the plant enzyme

Despite its importance in plant metabolism, no sequences of higher plant ATP-dependent phosphofructokinase (EC 2.7.1.11) are annotated in the databases. We have purified the enzyme from spinach leaves 309-fold to electrophoretic homogeneity. The purified enzyme was a homotetramer of approximately 52 kDa subunits with a specific activity of 600 mU x mg(-1) and a Km value for ATP of 81 microm. The purified enzyme was not activated by phosphate, but slightly inhibited instead, suggesting that it was the chloroplast isoform. The inclusion of adenosine 5'-(beta,gamma-imido)triphosphate was conducive to enzyme activity during the purification protocol. The sequences of eight tryptic peptides from the final protein preparation, which did not utilize pyrophosphate as a phosphoryl donor, were determined and an exactly corresponding cDNA was cloned. The sequence of enzymatically active spinach ATP-dependent phosphofructokinase suggests that a large family of genomics-derived higher plant sequences currently annotated in the databases as putative pyrophosphate-dependent phosphofructokinases according to sequence similarity is misannotated with respect to the cosubstrate.     

Pyrophosphate-dependent phosphofructokinase, an anaerobic glycolytic enzyme?

Recent evidence indicates that in as diverse organisms as unicellular eukaryotes, higher plants and prokaryotes, anaerobic glycolysis relies on a pyrophosphate-dependent phosphofructokinase instead of the classical ATP-dependent enzyme. This difference in phosphoryl donor specificity does not necessarily reflect a primitive metabolism, as thought earlier, but could rather be the result of convergent evolution, fostered by the energetic advantage conferred to the cell when glycolysis is the sole source of ATP.     

丙酮酸脱羧酶及其应用研究

丙酮酸脱羧酶(pyruvate decarboxylase,PDC),EC4.1.1.1,是一种胞内酶,是焦磷酸硫胺素(thiamine pyrophosphate,ThPP)依赖性的非氧化酶,是由辅酶ThPP、Mg2+和蛋白质构成的全酶,在辅助因子焦磷酸硫胺素和Mg2+参与下作用于丙酮酸而产生乙醛和CO2。PDC是丙酮酸合成乙醇的关键酶。它广泛存在于酵母菌、霉菌、细菌和植物等多种生物体中,不同来源的丙酮酸脱羧酶的结构、相对分子质量、酶学性质等均不尽相同。该文综述了丙酮酸脱羧酶生物学性质及其应用前景。     

Sequencing, expression, characterisation and phylogeny of the ADP-dependent phosphofructokinase from the hyperthermophilic, euryarchaeal Thermococcus zilligii

The full-length gene encoding the ADP-dependent phosphofructokinase (PFK) from the euryarchaeal Thermococcus zilligii was cloned, using degenerate primer polymerase chain reaction (PCR) combined with inverse-PCR techniques, and ultimately expressed in Escherichia coli. The expressed enzyme was biochemically characterised and found to be similar to the native enzyme for most properties examined. Sequence database searches suggest that this unique ADP-PFK possesses a limited phylogenetic distribution with homologues being found only in the other euryarchaeta Methanococcus jannaschii, Methanosarcina mazei and closely related members of the order Thermococcales. A phylogenetic analysis suggests that a single ancestral gene diverged to form the glucokinase and PFK lineages of this unique sequence family. Thus, the PFK reaction, one of the defining enzymatic activities of the Embden-Meyerhof pathway, can now be represented by three separate sequence families, the well-known PFKA family exemplified by the primary E. coli ATP-PFK (E.C. 2.7.1.11) and its associated ATP- and pyrophosphate-dependent PFKs (EC.2.7.1.90), the PFKB family (E. coli PFK 2 encoded by the pfkB gene and its homologues) and the ADP-PFKs of the Euryarchaeota reported here.     

Effect of ethylene treatment on the concentration of fructose-2,6-bisphosphate and on the activity of phosphofructokinase 2/fructose-2,6-bisphosphatase in banana

Preclimacteric bananas fruits were treated for 12 h with ethylene to induce the climacteric rise in respiration. One day after the end of the hormonal treatment, the two activities of the bifunctional enzyme, phosphofructokinase 2/fructose-2,6-bisphosphatase started to increase to reach fourfold their initial value 6 days later. By contrast, the activities of the pyrophosphate-dependent and of the ATP-dependent 6-phosphofructo-1-kinases remained constant during the whole experimental period, the first one being fourfold greater than the second. The concentrations of fructose 2,6-bisphosphate and of fructose 1,6-bisphosphate increased in parallel during 4 days and then slowly decreased, the second one being always about 100-fold greater than the first. The change in fructose 2,6-bisphosphate concentration can be partly explained by the rise of the bifunctional enzyme, but also by an early increase in the concentration of fructose 6-phosphate, the substrate of all phosphofructokinases, and also by the decrease in the concentration of glycerate 3-phosphate, a potent inhibitor of phosphofructokinase 2. The burst in fructose 2,6-bisphosphate and the activity of the pyrophosphate-dependent phosphofructokinase, which is in banana the only enzyme known to be sensitive to fructose 2,6-bisphosphate, can explain the well-known increase in fructose 1,6-bisphosphate which occurs during ripening.     

Presence of Prokaryotic and Eukaryotic Species in All Subgroups of the PPi-Dependent Group II Phosphofructokinase Protein Family

Inorganic pyrophosphate-dependent phosphofructokinase (PP(i)-PFK) of the amitochondriate eukaryote Mastigamoeba balamuthi was sequenced and showed about 60% identity to PP(i)-PFKs from two eubacteria, Propionibacterium freudenreichii and Sinorhizobium meliloti. These gene products represent a newly recognized lineage of PFKs. All four lineages of group II PFKs, as defined by phylogenetic analysis, contained both prokaryotic and eukaryotic species, underlining the complex evolutionary history of this enzyme.     

Mutations in sdh (succinate dehydrogenase genes) alter the thiamine requirement of Salmonella typhimurium.

Mutants lacking the first enzyme in de novo purine synthesis (PurF) can synthesize thiamine if increased levels of pantothenate are present in the culture medium (J. L. Enos-Berlage and D. M. Downs, J. Bacteriol. 178:1476-1479, 1996). Derivatives of purF mutants that no longer required pantothenate for thiamine-independent growth were isolated. Analysis of these mutants demonstrated that they were defective in succinate dehydrogenase (Sdh), an enzyme of the tricarboxylic acid cycle. Results of phenotypic analyses suggested that a defect in Sdh decreased the thiamine requirement of Salmonella typhimurium. This reduced requirement correlated with levels of succinyl-coenzyme A (succinyl-CoA), which is synthesized in a thiamine pyrophosphate-dependent reaction. The effect of succinyl-CoA on thiamine metabolism was distinct from the role of pantothenate in thiamine synthesis.     

pH dependence of the kinetic parameters for the pyrophosphate-dependent phosphofructokinase reaction supports a proton-shuttle mechanism

The pH dependence of kinetic parameters for the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii suggests that the enzyme catalyzes its reaction via general acid-base catalysis with the use of a proton shuttle. The base is required unprotonated in both reaction directions. In the direction of fructose 6-phosphate phosphorylation the base accepts a proton from the hydroxyl at C-1 of F6P and then donates it to protonate the leaving phosphate. Whether this occurs in one or two steps cannot be deduced from the present data. The maximum velocity of the reaction is pH independent in both reaction directions while V/K profiles exhibit pKs for binding groups (including enzyme and reactant functional groups) as well as pKs for enzyme catalytic groups. These data suggest that reactants bind only when correctly protonated and only to the correctly protonated form of the enzyme. Specifically, the requirement for two enzyme epsilon-amino groups in the protonated form for reactant binding was detected as was the requirement for the ionized phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, MgPPi and HPO4(2-). The protonation state of enzyme and reactant binding groups is in agreement with data obtained previously [Cho, Y.-K., & Cook, P. F. (1988) J. Biol. Chem. 263, 5135].     

Pyrophosphate-dependent phosphofructokinase. Conservation of protein sequence between the alpha- and beta-subunits and with the ATP-dependent phosphofructokinase

Full-length cDNA clones for the alpha- and beta-subunits of pyrophosphate-fructose 6-phosphate 1-phosphotransferase have been isolated from a cDNA expression library derived from potato tuber poly(A)+ RNA. The nucleotide sequences indicate that the alpha- and beta-subunits are related with about 40% of amino acid residues being identical. A comparison of the deduced amino acid sequences of both subunits of this enzyme with that of the major ATP-dependent fructose 6-phosphate 1-phosphotransferase from Escherichia coli (Shirakihara, Y., and Evans, P. R. (1988) J. Mol. Biol. 204, 973-994) showed little homology between the proteins except for regions involved in the binding of fructose 6-phosphate/fructose, 1,6-bisphosphate and possibly between regions binding pyrophosphate and the beta- and gamma-phosphates of ADP/ATP. A comparison of the derived secondary structures of the two subunits of the PPi-dependent enzyme with the known secondary structure of the E. coli ATP-dependent enzyme indicated that the overall structure of these enzymes is similar. These data suggest that catalytic activity resides on the beta-subunit of the pyrophosphate-dependent enzyme.     

Examination of intrinsic sulfonamide resistance in Bacillus anthracis: a novel assay for dihydropteroate synthase

Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPPi to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/alpha-glycerophosphate dehydrogenase reactions and monitors the disappearance of NADH at 340nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.     

Reconstitution into Liposomes of the Tonoplast ATP- and Pyrophosphate-Dependent Proton Pumps from Rubus hispidus Cell Cultures

The ATP- and pyrophosphate-dependent proton pumps from tonoplast-enriched vesicles prepared from Rubus hispidus cell cultures were solubilized in the presence of polyoxyethylene(9,10)p-t-octylphenol (Triton X-100) and reconstituted into liposomes of soybean phospholipids, using Bio-Beads SM-2 to remove the detergent. The specific activity of the two pumps was greatly increased by the solubilization-reconstitution procedure. Identical characteristics were found for pyrophosphate-dependent proton transport in native and reconstituted vesicles. On the other hand, the ATP-dependent proton transport of the reconstituted vesicles was no longer inhibited by KNO₃.