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.     

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.     

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.     

Kinetic analysis of PPi-dependent phosphofructokinase from Porphyromonas gingivalis

We have previously cloned the gene encoding a pyrophosphate-dependent phosphofructokinase (PFK), designated PgPFK, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease. In this study, recombinant PgPFK was purified to homogeneity, and biochemically characterized. The apparent K(m) value for fructose 6-phosphate was 2.2 mM, which was approximately 20 times higher than that for fructose 1,6-bisphosphate. The value was significantly greater than any other described PFKs, except for Amycolatopsis methanolica PFK which is proposed to function as a fructose 1,6 bisphosphatase (FBPase). The PgPFK appears to serves as FBPase in this organism. We postulate that this may lead to the gluconeogenic pathways to synthesize the lipopolysaccharides and/or glycoconjugates essential for cell viability.     

Characterisation of the ATP-dependent phosphofructokinase gene family from Arabidopsis thaliana

Plants possess two different types of phosphofructokinases, an ATP-dependent (PFK) and a pyrophosphate-dependent form (PFP). While plant PFPs have been investigated in detail, cDNA clones coding for PFK have not been identified in Arabidopsis thaliana. Searching the A. thaliana genome revealed 11 putative members of a phosphofructokinase gene family. Among those, four sequences showed high homology to the alpha- or beta-subunits of plant PFPs. Seven cDNAs resulted in elevated PFK, but not PFP activity after transient expression in tobacco leaves suggesting that they encode Arabidopsis PFKs. RT-PCR revealed different tissue-specific expression of the individual forms. Furthermore, analysis of GFP fusion proteins indicated their presence in different sub-cellular compartments.     

Expression, characterization, and crystallization of the pyrophosphate-dependent phosphofructo-1-kinase of Borrelia burgdorferi.

The two genes for the putative pyrophosphate-dependent phosphofructokinases (PPi-PFKs) of Borrelia burgdorferi were cloned by PCR and expressed in Escherichia coli, and their protein products were purified to near homogeneity. The larger of the two gene products, a 62-kDa protein, is an active PPi-PFK and exists in solution as a dimer. It has apparent K(m) values for fructose 6-P and PPi of 109 and 15 microM, respectively, and a pH optimum of 6.4 to 7.2. The 62-kDa protein was crystallized and subjected to preliminary diffraction analysis. The smaller gene product, a 48-kDa protein, exists in solution as a higher polymer and shows no ATP- or PPi-dependent activity, despite having a secondary structure as estimated by circular dichroism that is not significantly different from that of other PFKs.     

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.     

Inorganic pyrophosphate: fructose-6-phosphate 1-phosphotransferase of the potato tuber is related to the major ATP-dependent phosphofructokinase of E. coli

A procedure was developed for the purification of inorganic pyrophosphate: fructose-6-phosphate 1-phospho-transferase (PPi-PFK) from potato tubers. The enzyme has the structure alpha 4 beta 4 with a subunit of 68 kDa and a beta subunit of 60 kDa. The structural relationship of this enzyme to other PFKs and to fructose bisphosphatase was examined by immunoprecipitation and immunoblotting. Antibodies to the plant enzyme did not react with E. coli PFK. No cross-reaction was seen among the following enzymes or their antibodies: yeast fructose bisphosphatase; rabbit PFKs A, B, or the enzyme from brain; and the two subunits of the potato PPi-PFK. On the other hand, antibody to E. coli PFK-1 strongly cross-reacts with the 60 kDa polypeptide but not 68 kDa peptide.     

铜对梨形环棱螺抗氧化酶活性和金属硫蛋白含量的影响

本实验采用暴露重金属的方法,研究了不同浓度硫酸铜（Cu2+ 分别为0、0.005、0.01、0.02、0.05 mg/L）在不同暴露时间（0—14d）下对梨形环棱螺（Bellamya purificata）过氧化氢酶(CAT)、超氧化物歧化酶（SOD）、谷胱甘肽硫转移酶（GST）的活性、还原性谷胱甘肽（GSH）和金属硫蛋白（MT）含量的影响,以探讨Cu2+ 对梨形环棱螺的氧化损伤及其防御作用的机理,并为水环境Cu2+ 污染的早期诊断及生态风险评价提供科学的依据。结果表明：Cu2+对梨形环棱螺肝脏和鳃中CAT、SOD、GST、GSH 和MT 均有明显影响,表现出时间剂量效应。SOD在前4天、CAT在前3天酶活性总体上表现出诱导趋势, GST在前4天酶活性处于诱导状态,随着暴露时间的延长,酶活性下降,到第5天时表现出抑制趋势;随着时间的进一步增长,至14d时, 0.005 mg/L剂量组酶活性维持在正常值附近波动, 0.01 mg/L剂量组酶活性被诱导, 0.02 mg/L剂量组酶活性在肝脏中表现为诱导而在鳃中则被抑制,0.05 mg/L剂量组酶活性被抑制。肝脏和鳃GSH含量的变化与GST相似,在短时间内表现出诱导效应,肝脏GSH在暴露的前5天、鳃GSH在暴露的前4天均处于诱导状态,随着暴露时间的延长,0.005 mg/L剂量组表现出诱导,0.05 mg/L剂量组则受到抑制。MT在整个实验期间均处于诱导状态,各剂量组在0.5d被极显著诱导,随后MT含量出现起伏波动,有上升和下降,至第14天时达到一稳定水平。其中,0.01 mg/L剂量组肝脏的MT在整个实验期间均被极显著地诱导（P <0.01）,0.01 mg/L 剂量组的鳃组织MT除第10天外也被极显著诱导（P <0.01）。在暴露14d时,除0.05 mg/L剂量组的肝脏MT外,其余处于极显著诱导状态（P <0.01）。     

Paradoxical effects of copper and manganese on brain mitochondrial function

Defects in the mitochondrial genome have been associated with Parkinson's and Alzheimer's disease, and apoptosis can be triggered by the presence of energetically compromised mitochondria. Thus, in this study we have examined whether the divalent cations Cu2+ and Mn2+ could influence mitochondrial function in vitro. Mitochondrial electron transport was dose and time dependently reduced by Cu2+ to a greater extent with succinate as a substrate. Following a 60 min preincubation period, Mn2+ dose dependently inhibited electron transport to a greater extent with lactate and malate. In contrast, paradoxical effects were seen following a 5 min preincubation period with Mn2+. Cu2+ dose-dependently reduced NADH-dependent lactate dehydrogenase (LDH) activity, with almost complete inhibition apparent at 10 microM. An initial induction of LDH by 10 microM Mn2+ was partially reversed by higher concentrations of the metal. Cu2+ dose-dependently reduced flavin adenine dinucleotide (FAD)-dependent monoamine oxidase A (MAO-A) activity in a time-independent manner, with an IC50 value approximately 20 microM, whereas Mn2+ had no effect. In conclusion, it is proposed that Cu2+ and Mn2+ have differential effects on nicotinamide adenine dinucleotide (NAD) and FAD-dependent mitochondrial enzymes at the level of the essential cofactors. Cu2+ appears to exert an inhibitory effect on both NAD and FAD-dependent enzymes, but predominantly against the latter, including MAO-A and succinate dehydrogenase. The complex responses to Mn2+ may be due to dose-related effects on the interconversion of NAD and NADH and reversible enzymatic reactions employing this nucleotide cofactor.     

Putative pyrophosphate phosphofructose 1-kinase genes identified in sugar cane may be getting energy from pyrophosphate

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrus paradisi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the alpha and beta subunits of this enzyme. The deduced amino acid sequences showed 76 and 80% similarity with the corresponding alpha and beta subunits of C. paradisi. A high degree of similarity was also observed among the PFK b subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum. It appears that alpha and beta are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of b PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the b subunit of the pyrophosphate-dependent enzyme.     

Sequencing, cloning, and high-level expression of the pfp gene, encoding a PP(i)-dependent phosphofructokinase from the extremely thermophilic eubacterium Dictyoglomus thermophilum

The sequencing, cloning, and expression of the pfp gene from Dictyoglomus thermophilum, which consists of 1,041 bp and encodes a pyrophosphate-dependent phosphofructokinase, are described. A phylogenetic analysis indicates that the enzyme is closely related to the pyrophosphate-dependent enzyme from Thermoproteus tenax. The recombinant and native enzymes share a high degree of similarity for most properties examined.     

The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases

The pyrophosphate-dependent phosphofructokinase (PP_i-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5′ half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PP_i-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PP_i-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PP_i-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the α- and β-subunits of plant PP_i-PFKs. The third group (``X') containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group (``Y') comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PP_i-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers. Received: 5 December 1997 / Accepted: 18 March 1998     

Novel Zn2+ coordination by the regulatory N-terminus metal binding domain of Arabidopsis thaliana Zn(2+)-ATPase HMA2

Arabidopsis thaliana HMA2 is a Zn2+ transporting P1B-type ATPase required for maintaining plant metal homeostasis. HMA2 and all eukaryote Zn2+-ATPases have unique conserved N- and C-terminal sequences that differentiate them from other P1B-type ATPases. Homology modeling and structural comparison by circular dichroism indicate that the 75 amino acid long HMA2 N-terminus shares the betaalphabetabetaalpha folding present in most P1B-type ATPase N-terminal metal binding domains (N-MBDs). However, the characteristic metal binding sequence CysXXCys is replaced by Cys17CysXXGlu21, a sequence present in all plant Zn2+-ATPases. The isolated HMA2 N-MBD fragment binds a single Zn2+ (Kd 0.18 microM), Cd2+ (Kd 0.27 microM), or, with less affinity, Cu+ (Kd 13 microM). Mutagenesis studies indicate that Cys17, Cys18, and Glu21 participate in Zn2+ and Cd2+ coordination, while Cys17 and Glu21, but not Cys18, are required for Cu+ binding. Interestingly, the Glu21Cys mutation that generates a CysCysXXCys site is unable to bind Zn2+ or Cd2+ but it binds Cu+ with affinity (Kd 1 microM) higher than wild type N-MBD. Truncated HMA2 lacking the N-MBD showed reduced ATPase activity without significant changes in metal binding to transmembrane metal binding sites. Likewise, ATPase activity of HMA2 carrying mutations Cys17Ala, Cys18Ala, and Glu21Ala/Cys was also reduced but showed a metal dependence similar to the wild type enzyme. These observations suggest that plant Zn2+-ATPase N-MBDs have a folding and function similar to Cu+-ATPase N-MBDs. However, the unique Zn2+ coordination via two thiols and a carboxyl group provides selective binding of the activating metals to these regulatory domains. Metal binding through these side chains, although found in different sequences, appears as a common feature of both bacterial and eukaryotic Zn2+-ATPase N-MBDs.     

Highly selective blockade of the frog skin sodium channels by monovalent copper cations.

.5 mM Cu+ added to the mucosal side of frog skin caused rapid reversible inhibition of short-circuit current while no effect of Cu+ could be observed at the serosal side. In both cases Cu2+ was reduced to Cu+ by adding 10 mM ascorbic acid. Cu+ being similar to Na+ both in charge and crystal radius (0.096 and 0.095 nm, respectively) appears to block Na+ channels in the apical membrane. Cu2+ being of a smaller size (crystal radius 0.072 nm) was ineffective at the mucosal side causing only a rather slow irreversible inhibition of Na+ transport when added to the serosal bathing solution.     

Copper induces histone hypoacetylation through directly inhibiting histone acetyltransferase activity

The abnormal accumulation of Cu2+ is closely correlated with the incidence of different diseases, such as Alzheimer's disease and Wilson disease. To study in vivo functions of Cu2+ will lead to a better understanding of the nature of these diseases. In the present study, effect of Cu2+ on histone acetylation was investigated in human hepatoma cells. Exposure of cells to Cu2+ resulted in a significant decrease of histone acetylation, as indicated by the decrease of the overall histone acetylation and the decrease of histone H3 and H4 acetylation. Since histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlled the state of histone acetylation in vivo, we tested their contribution to the inhibition of Cu2+ on histone acetylation. One hundred nanomolar trichostatin A, the specific inhibitor of HDAC, did not attenuate the inhibitory effect of Cu2+ on histone acetylation. Combined with that Cu2+ showed no effect on the in vitro activity of HDAC, these results led to the conclusion that it is HAT, but not HDAC that is involved in Cu2+ -induced histone hypoacetylation. This conclusion was confirmed by the facts that (1) Cu2+ significantly inhibited the in vitro activity of HAT, (2) Cu2+ -treated cells possessed a lower HAT activity than control cells, and (3) 50 or 100 microM bathocuproine disulfonate, a chelator of Cu2+, significantly attenuated the inhibition of Cu2+ on HAT activity and histone acetylation in the similar pattern. Combined with that Cu2+ showed no or obvious cytotoxicity at 100 or 200 microM in human hepatoma cells, and the previous study that Cu2+ inhibits the histone H4 acetylation of yeast cells at nontoxic or toxic levels, the data presented here suggest that inhibiting histone acetylation is probably one general in vivo function of Cu2+, where HAT is its molecular target.     

Accumulation of copper by roots,hypocotyls, cotyledons and leaves of sunflower (Helianthus annuus L.)

The effects of different concentrations of copper sulfate on the growth of and the accumulation of Cu2+ by root, hypocotyl, cotyledon and leaf growth of sunflower (Helianthus annuus L.) were examined in this study. The concentrations of copper sulfate (CuSO4 x 5H2O) used were in the range from 10(-5) to 10(-3) M. Seedlings exposed to 10(-5) M Cu2+ solution exhibited a 33% increase in growth (P < 0.005) when compared with the root length of the control. The seedlings treated with 10(-3) M Cu2+ were significantly inhibited in shoot growth (P < 0.005). The Cu2+ content in roots, hypocotyls, cotyledons and leaves increased with increasing solution Cu2+ concentration. The roots of plants exposed to 10(-3) M Cu2+ accumulated a large amount of Cu (1070 microgram/g DW), and the Cu2+ level was approximately 25 fold higher than that of control. The Cu2+ contents in sunflower roots treated with 10(-4) and 10(-5) M Cu2+ were about 3.3 and 2.6 fold higher than the control, respectively. Also, the Cu2- level of the roots exposed to 10(-3) M Cu2+ was approximately 7.7 and 9.8 fold respectively, in comparison with the roots of plants grown in 10(-4) and 10(-5) M Cu2+. At 10(-3) M Cu2+, the Cu accumulated mainly in the roots (about 73%), and small amounts of Cu2+ (27%) were translocated to the hypocotyls, cotyledons and leaves. The Cu2+ concentration in the roots was less than that of the above parts of seedlings in treated groups with 10(-5) - 10(-4) M Cu2+. H. annuus has potential ability to accumulate Cu without being overly sensitive to Cu toxicity.     

Reinvestigation of metal ion specificity for quinone cofactor biogenesis in bacterial copper amine oxidase

The topa quinone (TPQ) cofactor of copper amine oxidase is generated by copper-assisted self-processing of the precursor protein. Metal ion specificity for TPQ biogenesis has been reinvestigated with the recombinant phenylethylamine oxidase from Arthrobacter globiformis. Besides Cu2+ ion, some divalent metal ions such as Co2+, Ni2+, and Zn2+ were also bound to the metal site of the apoenzyme so tightly that they were not replaced by excess Cu2+ ions added subsequently. Although these noncupric metal ions could not initiate TPQ formation under the atmospheric conditions, we observed slow spectral changes in the enzyme bound with Co2+ or Ni2+ ion under the dioxygen-saturating conditions. Resonance Raman spectroscopy and titration with phenylhydrazine provided unambiguous evidence for TPQ formation by Co2+ and Ni2+ ions. Steady-state kinetic analysis showed that the enzymes activated by Co2+ and Ni2+ ions were indistinguishable from the corresponding metal-substituted enzymes prepared from the native copper enzyme (Kishishita, S., Okajima, T., Kim, M., Yamaguchi, H., Hirota, S., Suzuki, S., Kuroda, S., Tanizawa, K., and Mure, M. (2003) J. Am. Chem. Soc. 125, 1041-1055). X-ray crystallographic analysis has also revealed structural identity of the active sites of Co- and Ni-activated enzymes with Cu-enzyme. Thus Cu2+ ion is not the sole metal ion assisting TPQ formation. Co2+ and Ni2+ ions are also capable of forming TPQ, though much less efficiently than Cu2+.