Search

Giardia lamblia is a single-celled eukaryotic parasite with a small genome and is considered an early divergent eukaryote. The pentose phosphate pathway (PPP) plays an essential role in the oxidative stress defense of the parasite and the production of ribose-5-phosphate. In this parasite, the glucose-6-phosphate dehydrogenase (G6PD) is fused with the 6-phosphogluconolactonase (6PGL) enzyme, generating the enzyme named G6PD::6PGL that catalyzes the first two steps of the PPP. Here, we report that the G6PD::6PGL is a bifunctional enzyme with two catalytically active sites. We performed the kinetic characterization of both domains in the fused G6PD::6PGL enzyme, as well as the individual cloned G6PD. The results suggest that the catalytic activity of G6PD and 6PGL domains in the G6PD::6PGL enzyme are more efficient than the individual proteins. Additionally, using enzymatic and mass spectrometry assays, we found that the final metabolites of the catalytic reaction of the G6PD::6PGL are 6-phosphoglucono-δ-lactone and 6-phosphogluconate. Finally, we propose the reaction mechanism in which the G6PD domain performs the catalysis, releasing 6-phosphoglucono-δ-lactone to the reaction medium. Then, this metabolite binds to the 6PGL domain catalyzing the hydrolysis reaction and generating 6-phosphogluconate. The structural difference between the G. lamblia fused enzyme G6PD::6PGL with the human G6PD indicate that the G6PD::6PGL is a potential drug target for the rational synthesis of novels anti-Giardia drugs.

Recent work revealed that PGD2, an Arabidopsis 6-phosphogluconate dehydrogenase (6-PGD) catalysing the third step of the oxidative pentose-phosphate pathway (OPPP) in peroxisomes, is essential during fertilization. Earlier studies on the second step, catalysed by PGL3, a dually targeted Arabidopsis 6-phosphogluconolactonase (6-PGL), reported the importance of OPPP reactions in plastids but their irrelevance in peroxisomes. Assuming redundancy of 6-PGL activity in peroxisomes, we examined the sequences of other higher plant enzymes. In tomato, there exist two 6-PGL isoforms with the strong PTS1 motif SKL. However, their analysis revealed problems regarding peroxisomal targeting: reporter–PGL detection in peroxisomes required construct modification, which was also applied to the Arabidopsis isoforms. The relative contribution of PGL3 versus PGL5 during fertilization was assessed by mutant crosses. Reduced transmission ratios were found for pgl3-1 (T-DNA-eliminated PTS1) and also for knock-out allele pgl5-2. The prominent role of PGL3 showed as compromised growth of pgl3-1 seedlings on sucrose and higher activity of mutant PGL3-1 versus PGL5 using purified recombinant proteins. Evidence for PTS1-independent uptake was found for PGL3-1 and other Arabidopsis PGL isoforms, indicating that peroxisome import may be supported by a piggybacking mechanism. Thus, multiple redundancy at the level of the second OPPP step in peroxisomes explains the occurrence of pgl3-1 mutant plants.

IntroductionThe pentose phosphate pathway (PPP) is essential for NADPH generation and redox homeostasis in cancer, including glioblastomas. However, the precise contribution to redox and tumor proliferation of the second PPP enzyme 6-phosphogluconolactonase (PGLS), which converts 6-phospho-δ-gluconolactone to 6-phosphogluconate (6PG), remains unclear. Furthermore, non-invasive methods of assessing PGLS activity are lacking. The goal of this study was to examine the role of PGLS in glioblastomas and assess the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive imaging.MethodsTo interrogate the function of PGLS in redox, PGLS expression was silenced in U87, U251 and GS2 glioblastoma cells by RNA interference and levels of NADPH and reduced glutathione (GSH) measured. Clonogenicity assays were used to assess the effect of PGLS silencing on glioblastoma proliferation. Hyperpolarized δ-[1-13C]gluconolactone metabolism to 6PG was assessed in live cells treated with the chemotherapeutic agent temozolomide (TMZ) or with vehicle control. 13C 2D echo-planar spectroscopic imaging (EPSI) studies of hyperpolarized δ-[1-13C]gluconolactone metabolism were performed on rats bearing orthotopic glioblastoma tumors or tumor-free controls on a 3T spectrometer. Longitudinal 2D EPSI studies of hyperpolarized δ-[1-13C]gluconolactone metabolism and T2-weighted magnetic resonance imaging (MRI) were performed in rats bearing orthotopic U251 tumors following treatment with TMZ to examine the ability of hyperpolarized δ-[1-13C]gluconolactone to report on treatment response.ResultsPGLS knockdown downregulated NADPH and GSH, elevated oxidative stress and inhibited clonogenicity in all models. Conversely, PGLS expression and activity and steady-state NADPH and GSH were higher in tumor tissues from rats bearing orthotopic glioblastoma xenografts relative to contralateral brain and tumor-free brain. Importantly, [1-13C]6PG production from hyperpolarized δ-[1-13C]gluconolactone was observed in live glioblastoma cells and was significantly reduced by treatment with TMZ. Furthermore, hyperpolarized δ-[1-13C]gluconolactone metabolism to [1-13C]6PG could differentiate tumor from contralateral normal brain in vivo. Notably, TMZ significantly reduced 6PG production from hyperpolarized δ-[1-13C]gluconolactone at an early timepoint prior to volumetric alterations as assessed by anatomical imaging.ConclusionsCollectively, we have, for the first time, identified a role for PGLS activity in glioblastoma proliferation and validated the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive in vivo imaging of glioblastomas and their response to therapy.

Malaria is a parasitic disease caused by Plasmodium spp., being one of the major causes of death worldwide with two-hundred million new infections and hundreds of thousands of deaths in 2015. Despite the important advances in its prevention and treatment, its resistance to current drug therapies is still a serious risk in its eradication. There is urgency in finding novel targets and drugs operating by novel mechanisms, avoiding cross-resistance to classical antimalarials. In this context, the bifunctional enzyme Glucose-6- phosphate dehydrogenase 6-phosphogluconolactonase appears to be a promising therapeutic target due to its crucial role in regulating the PPP pathway (pentose phosphate pathway), which is the major source of redox potential in Plasmodium falciparum. In the last few years, our group detected a specific mutation between the human and the Plasmodium falciparum form in the binding site of Glucose-6-phosphate (G6P), the endogenous ligand of Glucose-6-phosphate dehydrogenase (G6PD). This mutation involves the substitution of an Arginine (human) by an Aspartate (parasite), which allowed us to create a validated in-house homology model of PfG6PD. Based on this result, the group has focused their efforts, through different molecular modelling techniques, in the discovery of selective scaffolds against PfG6PD. Current efforts address the development of a complete structural model of the bifunctional enzyme, which may offer novel opportunities to develop molecules capable of inhibiting this relevant enzyme.

Background Since malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites. Methods Plasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium. Results Here the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher KM values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to also be active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity. Conclusion The characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites’ redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up. Electronic supplementary material The online version of this article (10.1186/s12936-019-2651-z) contains supplementary material, which is available to authorized users.

The malarial parasite Plasmodium falciparum is exposed to substantial redox challenges during its complex life cycle. In intraerythrocytic parasites, haemoglobin breakdown is a major source of reactive oxygen species. Deficiencies in human glucose-6-phosphate dehydrogenase, the initial enzyme in the pentose phosphate pathway (PPP), lead to a disturbed redox equilibrium in infected erythrocytes and partial protection against severe malaria. In P. falciparum, the first two reactions of the PPP are catalysed by the bifunctional enzyme glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase (PfGluPho). This enzyme differs structurally from its human counterparts and represents a potential target for drugs. In the present study we used epitope tagging of endogenous PfGluPho to verify that the enzyme localises to the parasite cytosol. Furthermore, attempted double crossover disruption of the PfGluPho gene indicates that the enzyme is essential for the growth of blood stage parasites. As a further step towards targeting PfGluPho pharmacologically, ellagic acid was characterised as a potent PfGluPho inhibitor with an IC50 of 76 nM. Interestingly, pro-oxidative drugs or treatment of the parasites with H2O2 only slightly altered PfGluPho expression or activity under the conditions tested. Furthermore, metabolic profiling suggested that pro-oxidative drugs do not significantly perturb the abundance of PPP intermediates. These data indicate that PfGluPho is essential in asexual parasites, but that the oxidative arm of the PPP is not strongly regulated in response to oxidative challenge.

A high-throughput screen of the NIH’s MLSMR collection of ∼340000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfG6PD). PfG6PD is important for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human orthologue. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. In P. falciparum, the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood cells rely on accelerated glucose flux, they depend on the G6PD activity of PfGluPho. The lead compound identified from this effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-be...

Plasmodium falciparum causes severe malaria infections in millions of people every year. The parasite is developing resistance to the most common antimalarial drugs, which creates an urgent need for new therapeutics. A promising and attractive target for antimalarial drug design is the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) of P. falciparum, which catalyzes the key step in the parasites’ pentose phosphate pathway. In this study we describe the development of a high-throughput screening assay to identify small-molecule inhibitors of recombinant PfGluPho. The optimized assay was used to screen three small-molecule compound libraries, namely LOPAC(™) (Sigma-Aldrich, 1,280 compounds), Spectrum(™) (Microsource discovery system, 1,969 compounds), and DIVERSet(™) (ChemBridge, 49,971 compounds). These pilot screens identified 899 compounds that inhibited PfGluPho activity by at least 50%. Selected compounds were further studied to determine IC(50) values in an orthogonal assay, the type of inhibition and reversibility, and effects on P. falciparum growth. Screening results and follow-up studies for selected PfGluPho inhibitors are presented. Our high-throughput screening assay may provide the basis to identify novel and urgently needed antimalarial drugs.

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.

Infection with wild-type Listeria monocytogenes activates a host cytosolic surveillance response characterized by the expression of beta interferon (IFN-β). We performed a genetic screen to identify L. monocytogenes transposon insertion mutants that induced altered levels of host IFN-β expression. One mutant from this screen induced elevated levels of IFN-β and harbored a Tn 917 insertion upstream of lmo0558. This study identified lmo0558 as the 6-phosphogluconolactonase gene ( pgl ), which encodes the second enzyme in the pentose phosphate pathway. pgl mutant L. monocytogenes accumulated and secreted large amounts of gluconate, likely derived from labile 6-phosphogluconolactone, the substrate of Pgl. The pgl deletion mutant had decreased growth in glucose-limiting minimal medium but grew normally when excess glucose was added. Microarray analysis revealed that the pgl deletion mutant had increased expression of several β-glucosidases, consistent with known inhibition of β-glucosidases by 6-phosphogluconolactone. While growth in macrophages was indistinguishable from that of wild-type bacteria, pgl mutant L. monocytogenes exhibited a 15- to 30-fold defect in growth in vivo. In addition, L. monocytogenes harboring an in-frame deletion of pgl was more sensitive to oxidative stress. This study identified L. monocytogenes pgl and provided the first link between the bacterial pentose phosphate pathway and activation of host IFN-β expression.

The bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (G6PD-6PGL) found in Plasmodium falciparum has unique structural and functional characteristics restricted to this genus. This study was designed to examine the effects of RNA-mediated PfG6PD-6PGL gene silencing in cultures of P. falciparum on the expression of parasite antioxidant defense genes at the transcription level. The highest degree of G6PD-6PGL silencing achieved was 86% at the mRNA level, with a recovery to almost normal levels within 24 h, indicating only transient diminished expression of the PfG6PD-6PGL gene. PfG6PD-6PGL silencing caused arrest of the trophozoite stage and enhanced gametocyte formation. In addition, an immediate transcriptional response was shown by thioredoxin reductase suggesting that P. falciparum G6PD-6PGL plays a physiological role in the specific response of the parasite to intracellullar oxidative stress. P. falciparum transfection with an empty DNA vector also promoted intracellular stress, as determined by mRNA up-regulation of antioxidant genes. Collectively, our findings point to an important role for this enzyme in the parasite's infection cycle. The different characteristics of G6PD-6PGL with respect to its homologue in the host make it an ideal target for therapeutic strategies.

The pentose-phosphate pathway (PPP) is an important part of central metabolism in many organisms. A pgl− mutation that decreases the efficiency of the second stage of PPP has been described and mapped at approx. 17.2 min of the Escherichia coli chromosome more than 30 years ago. Although it has recently been shown that deletion of ORF ybhE leads to earlier detected Pgl− phenotype for E. coli mutant strain, 6-phosphogluconolactonase from this organism has not been characterized. In the present, independent investigation we show that the Pgl− phenotype of ΔybhE MG1655 could be complemented by insertion of the well-characterized pgl gene from Pseudomonas putida whose protein product has no visible homology with E. coli YbhE. Moreover, a final confirmation that ybhE actually encodes 6PGL in E. coli was obtained through overexpression of the cloned gene, purification of the protein product, followed by direct determination of its enzymatic activity in vitro.

We report identification of the Escherichia coli ybhE gene as the pgl gene that encodes 6-phosphogluconolactonase. A tentative identification was first made based on the known approximate location of the pgl gene and the similarity of the presumptive ybhE-encoded protein sequence to a known Pgl enzyme. To test this notion, the ybhE gene was deleted and replaced with a drug marker. Like previously characterized pgl mutants, the ybhE deletion mutant had a Blu − phenotype (dark-blue staining with iodine due to accumulation of starch after growth on minimal maltose) and demonstrated impaired growth on minimal glucose medium when combined with a pgi mutation. Biochemical assay of crude extracts for 6-phosphogluconolactonase enzymatic activity showed that ybhE encodes this activity. The ybhE gene was transferred from the E. coli chromosome to an expression vector. This ybhE clone complemented both the precise deletion of the ybhE gene and a larger deletion, pgl Δ 8 , for the Blu − phenotype and for phosphogluconolactonase activity, confirming that ybhE is the pgl gene. A newly observed phenotype of pgl strains is a lowered frequency of appearance of Bgl + mutants that can utilize the β-glucoside salicin. This is likely due to poor growth of Bgl + pgl strains on salicin due to the accumulation of 6-phosphogluconolactone.

Summary. As the production of NADPH in the pentose phosphate pathway is the main antioxidant defence mechanism available to the Plasmodium falciparum, we have studied the expression of P. falciparum glucose 6-phosphate dehydrogenase-6-phosphogluconolactonase (PfG6PD-6PGL) in G6PD-deficient and normal erythrocyte host cells. Both erythrocytes infected in vitro with a laboratory isolate and erythrocytes from natural human infections were used. Total RNA was prepared from parasites collected from five G6PD-deficient and nine G6PD-normal children in Ibadan, Nigeria, selected after screening 189 rural schoolchildren and 68 clinical malaria patients, and was subjected to Northern blot analysis. The probe was a cDNA fragment of the G6PD domain of the PfG6PD-6PGL gene, with an internal control probe of P. falciparum 18S ribosomal RNA. Quantification was performed using a phosphoimager. Relative to internal control, the abundance of PfG6PD-6PGL mRNA (mean ± standard deviation) was lower in parasites from G6PD-deficient children (0·29 ± 0·27) than in G6PD-normal control subjects (0·74 ± 0·26) (P = 0·014, Mann–Whitney U-test). Although confirmation in a larger study is required, our results suggest a lower relative abundance of PfG6PD-6PGL, and presumably antioxidant activity, in malaria parasites from G6PD-deficient hosts, thus extending the current knowledge of the mechanism of G6PD-deficiency related host protection.

Trypanosomatids are parasitic protists that have part of their glycolytic pathway sequestered inside peroxisome-like organelles: the glycosomes. So far, at least one enzyme of the pentose-phosphate pathway has been found to be associated partially with glycosomes. Here, we describe how two genes from Trypanosoma brucei, coding for the first two enzymes of the pentose-phosphate pathway, i.e. glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase, were identified by in silico screening of trypanosome genome project data bases. These genes were cloned and sequenced. Analysis of the lactonase sequence revealed that it contained a C-terminal peroxisome targeting signal in agreement with its subcellular localization in the bloodstream form trypanosome (15% glycosomal and 85% cytosolic). However, the dehydrogenase sequence did not reveal any targeting signal, despite its localization inside glycosomes. The corresponding enzymes have been overexpressed in Escherichia coli and purified, and their biochemical characteristics have been determined.

We report the sequence of a human cDNA encoding a protein homologous to devB (a bacterial gene often found in proximity to the gene encoding glucose-6-phosphate dehydrogenase in bacterial genomes) and to the C-terminal part of human hexose-6-phosphate dehydrogenase. The protein was expressed in Escherichia coli, purified and shown to be 6-phosphogluconolactonase, the enzyme catalyzing the second step of the pentose phosphate pathway. Sequence analysis indicates that bacterial devB proteins, the C-terminal part of hexose-6-phosphate dehydrogenase and yeast Sol1-4 proteins are most likely also 6-phosphogluconolactonases and that these proteins are related to glucosamine-6-phosphate isomerases.

Supplemental Figures, Tables, and AppendicesxDownload (3.04 MB ) Supplemental Figures, Tables, and Appendices

Sugar metabolism and the oxidative pentose phosphate pathway (OPPP) are strongly implicated in N assimilation, although the relationship between them and the roles of the plastidial and cytosolic OPPP have not been established genetically. We studied a knock-down mutant of the plastid-localized OPPP enzyme 6-phosphogluconolactonase 3 (PGL3). pgl3-1 plants exhibited relatively greater resource allocation to roots but were smaller than the wild type. They had a lower content of amino acids and free NO – 3 in leaves than the wild type, despite exhibiting comparable photosynthetic rates and efficiency, and normal levels of many other primary metabolites. When N-deprived plants were fed via the roots with 15 NO – 3 , pgl3-1 exhibited normal induction of OPPP and nitrate assimilation genes in roots, and amino acids in roots and shoots were labeled with 15N at least as rapidly as in the wild type. However, when N-replete plants were fed via the roots with sucrose, expression of specific OPPP and N assimilation genes in roots increased in the wild type but not in pgl3-1 . Thus, sugar-dependent expression of N assimilation genes requires OPPP activity and the specificity of the effect of the pgl3-1 mutation on N assimilation genes establishes that it is not the result of general energy deficiency or accumulation of toxic intermediates. We conclude that expression of specific nitrate assimilation genes in the nucleus of root cells is positively regulated by a signal emanating from OPPP activity in the plastid.

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based.

International audience; Trypanosoma brucei is the causative agent of African sleeping sickness. Current work for the development of new drugs against this pathology includes evaluation of enzymes of the pentose phosphate pathway (PPP), which first requires a clear understanding of their function and mechanism of action. In this context, we focused on T. brucei 6-phosphogluconolactonase (Tb6PGL), which converts delta-6-phosphogluconolactone into 6-phosphogluconic acid in the second step of the PPP. We have determined the crystal structure of Tb6PGL in complex with two ligands, 6-phosphogluconic acid and citrate, at 2.2 A and 2.0 A resolution, respectively. We have performed molecular dynamics (MD) simulations on Tb6PGL in its empty form and in complex with delta-6-phosphogluconolactone, its natural ligand. Analysis of the structural data and MD simulations allowed us to propose a detailed enzymatic mechanism for 6PGL enzymes.

International audience; Enzymes from the pentose phosphate pathway (PPP) are potential drug targets for the development of new drugs against Trypanosoma brucei, the causative agent of African sleeping disease: for instance, the 6-phosphogluconate dehydrogenase is currently studied actively for such purposes. Structural and functional studies are necessary to better characterize the associated enzymes and compare them to their human homologues, in order to undertake structure-based drug design studies on such targets. In this context, the crystal structure of 6-phosphogluconolactonase (6PGL) from T. brucei, the second enzyme from PPP, was determined at 2.1 Å resolution. Comparison of its sequence and structure to other related proteins in the 6PGL family with a known structure (Thermotoga maritima Tm6GPL 1PBT and Vibrio cholerae Vc6PGL (1Y89), which have not been discussed in print), or in the glucosamine-6-phosphate-deaminase family (hexameric Escherichia coli 1DEA and monomeric Bacillus subtilis 2BKV), allowed the identification of the 6PGL active site. In addition to the analysis of the crystal structure, 3D NMR interaction studies and docking experiments are reported here. Key residues involved in substrate binding or in catalysis were identified.

The functional coupling of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase was investigated in rat liver microsomal vesicles. The activity of both enzymes was latent in intact vesicles, indicating the intraluminal localization of their active sites. Glucose-6-phosphate, a substrate for hexose-6-phosphate dehydrogenase, stimulated the cortisone reductase activity of 11beta-hydroxysteroid dehydrogenase type 1. Inhibition of glucose-6-phosphate uptake by S3483, a specific inhibitor of the microsomal glucose-6-phosphate transporter, decreased this effect. Similarly, cortisone increased the intravesicular accumulation of radioactivity upon the addition of radiolabeled glucose-6-phosphate, indicating the stimulation of hexose-6-phosphate dehydrogenase activity. A correlation was shown between glucose-6-phosphate-dependent cortisone reduction and cortisone-dependent glucose-6-phosphate oxidation. The results demonstrate a close cooperation of the enzymes based on co-localization and the mutual generation of cofactors for each other.

Plasmodium berghei glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (G6PD-6PGL) is a bifunctional enzyme with significant sequence similarity in both the 6PGL and G6PD domains to the Plasmodium falciparum enzyme. A recombinant form of the P. berghei enzyme was found to have both G6PD and 6PGL activities, and therefore catalyses the first two steps in the pentose phosphate pathway. Genes encoding very similar proteins are also found in three other malarial parasites, Plasmodium yoelii, Plasmodium chabaudi and Plasmodium knowlesi. All of these predicted enzymes contain unique parasite insertions in corresponding positions in the G6PD domain but the insertions differ in size and sequence. Such insertions are a common feature of malarial proteins but their origin and function is unknown. Excision of the insertion sequence in the P. berghei protein renders the G6PD domain inactive, although the 6PGL activity is unaffected. Replacing the insertion sequence in P. berghei with the insertion sequence from P. falciparum restores some of the G6PD activity and also enhances 6PGL activity. We conclude that although the insertions are evolving rapidly they have an essential role in the activity of the bifunctional enzyme.

The pentose-phosphate pathway provides reductive power and nucleotide precursors to the cell through oxidative and nonoxidative branches, respectively. 6-Phosphogluconolactonase is the second enzyme of the oxidative branch and catalyzes the hydrolysis of 6-phosphogluconolactones, the products of glucose 6-phosphate oxidation by glucose-6-phosphate dehydrogenase. The role of 6-phosphogluconolactonase was still questionable, because 6-phosphogluconolactones were believed to undergo rapid spontaneous hydrolysis. In this work, nuclear magnetic resonance spectroscopy was used to characterize the chemical scheme and kinetic features of the oxidative branch. We show that 6-phosphogluconolactones have in fact a nonnegligible lifetime and are highly electrophilic compounds. The delta form (1-5) of the lactone is the only product of glucose 6-phosphate oxidation. Subsequently, it leads to the gamma form (1-4) by intramolecular rearrangement. However, only the delta form undergoes spontaneous hydrolysis, the gamma form being a "dead end" of this branch. The delta form is the only substrate for 6-phosphogluconolactonase. Therefore, 6-phosphogluconolactonase activity accelerates hydrolysis of the delta form, thus preventing its conversion into the gamma form. Furthermore, 6-phosphogluconolactonase guards against the accumulation of delta-6-phosphogluconolactone, which may be toxic through its reaction with endogenous cellular nucleophiles. Finally, the difference between activity of human, Trypanosoma brucei, and Plasmodium falciparum 6-phosphogluconolactonases is reported and discussed.

Plasmodium falciparum glucose 6-phosphate dehydrogenase (Pf Glc6PD), compared to other Glc6PDs has an additional 300 amino acids at the N-terminus. They are not related to Glc6PD but are similar to a family of proteins (devb) of unknown function, some of which are encoded next to Glc6PD in certain bacteria. The human devb homologue has recently been shown to have 6-phosphogluconolactonase (6PGL) activity. This suggests Pf Glc6PD may be a bifunctional enzyme, the evolution of which has involved the fusion of adjacent genes. Further functional analysis of Pf Glc6PD has been hampered because parts of the gene could not be cloned. We have isolated and sequenced the corresponding Plasmodium berghei gene and shown it encodes an enzyme (Pb Glc6PD) with the same structure as the P. falciparum enzyme. Pb Glc6PD is 950 amino acids long with significant sequence similarity in both the devb and Glc6PD domains with the P. falciparum enzyme. The P. berghei enzyme does not have an asparagine-rich segment between the N and C halves and it contains an insertion at the same point in the Glc6PD region as the P. falciparum enzyme but the insertion in the P. berghei is longer (110 versus 62 amino acids) and unrelated in sequence to the P. falciparum insertion. Though expression of this enzyme in bacteria produced largely insoluble protein, conditions were found where the full-length enzyme was produced in a soluble form which was purified via a histidine tag. We show that this enzyme has both Glc6PD and 6PGL activities. Thus the first two steps of the pentose phosphate pathway are catalysed by a single novel bifunctional enzyme in these parasites.

A cyclic version of the Entner-Doudoroff pathway is used by Pseudomonas aeruginosa to metabolize carbohydrates. Genes encoding the enzymes that catabolize intracellular glucose to pyruvate and glyceraldehyde 3-phosphate are coordinately regulated, clustered at 39 min on the chromosome, and collectively form the hex regulon. Within the hex cluster is an open reading frame (ORF) with homology to the devB/SOL family of unidentified proteins. This ORF encodes a protein of either 243 or 238 amino acids; it overlaps the 5′ end of zwf (encodes glucose-6-phosphate dehydrogenase) and is followed immediately by eda (encodes the Entner-Doudoroff aldolase). The devB/SOL homolog was inactivated in P. aeruginosa PAO1 by recombination with a suicide plasmid containing an interrupted copy of the gene, creating mutant strain PAO8029. PAO8029 grows at 9% of the wild-type rate using mannitol as the carbon source and at 50% of the wild-type rate using gluconate as the carbon source. Cell extracts of PAO8029 were specifically deficient in 6-phosphogluconolactonase (Pgl) activity. The cloned devB/SOL homolog complemented PAO8029 to restore normal growth on mannitol and gluconate and restored Pgl activity. Hence, we have identified this gene as pgl and propose that the devB/SOL family members encode 6-phosphogluconolactonases. Interestingly, three eukaryotic glucose-6-phosphate dehydrogenase (G6PDH) isozymes, from human, rabbit, and Plasmodium falciparum , contain Pgl domains, suggesting that the sequential reactions of G6PDH and Pgl are incorporated in a single protein. 6-Phosphogluconolactonase activity is induced in P. aeruginosa PAO1 by growth on mannitol and repressed by growth on succinate, and it is expressed constitutively in P. aeruginosa PAO8026 ( hexR ). Taken together, these results establish that Pgl is an essential enzyme of the cyclic Entner-Doudoroff pathway encoded by pgl , a structural gene of the hex regulon.