As the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH) is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG). Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications.
The crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1) from Saccharomyces cerevisiae has been determined at 2.37 Å resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-α helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 ± 9 μM for 6-phosphogluconate and of 35 ± 6 μM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution.
The overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not necessary for the dimerization. This domain also works as a lid on the substrate binding pocket to control the binding of substrate and the release of product, so it is indispensable for the 6PGDH activity. Moreover, the co-crystallized citrate molecules, which mimic the binding mode of the substrate 6-phosphogluconate, provided us a novel strategy to design the 6PDGH inhibitors.
The crystal structure of the prephenate dehydrogenase component of the bifunctional H. influenzae TyrA reveals unique structural differences between bifunctional and monofunctional TyrA enzymes.
Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P)+-dependent oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD+ has been determined to 2.0 Å resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal α/β dinucleotide-binding domain and a C-terminal α-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an α–β motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP+ and NAD+. The loop between β5 and β6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from the loop between β5 and β6) and Arg365′ (from the additional C-terminal helix of the adjacent monomer) is observed that might be involved in gating the active site.
tyrosine biosynthesis; prephenate; chorismate; Haemophilus influenzae; structural genomics
Sequence analysis of a 6.3-kbp genomic EcoRI-fragment of Alcaligenes eutrophus, which was recently identified by using a dihydrolipoamide dehydrogenase-specific DNA probe (A. Pries, S. Hein, and A. Steinbüchel, FEMS Microbiol. Lett. 97:227-234, 1992), and of an adjacent 1.0-kbp EcoRI fragment revealed the structural genes of the A. eutrophus pyruvate dehydrogenase complex, pdhA (2,685 bp), pdhB (1,659 bp), and pdhL (1,782 bp), encoding the pyruvate dehydrogenase (E1), the dihydrolipoamide acetyltransferase (E2), and the dihydrolipoamide dehydrogenase (E3) components, respectively. Together with a 675-bp open reading frame (ORF3), the function of which remained unknown, these genes occur colinearly in one gene cluster in the order pdhA, pdhB, ORF3, and pdhL. The A. eutrophus pdhA, pdhB, and pdhL gene products exhibited significant homologies to the E1, E2, and E3 components, respectively, of the pyruvate dehydrogenase complexes of Escherichia coli and other organisms. Heterologous expression of pdhA, pdhB, and pdhL in E. coli K38(pGP1-2) and in the aceEF deletion mutant E. coli YYC202 was demonstrated by the occurrence of radiolabeled proteins in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively. A three-step procedure using chromatography on DEAE-Sephacel, chromatography on the triazine dye affinity medium Procion Blue H-ERD, and heat precipitation purified the E3 component of the A. eutrophus pyruvate dehydrogenase complex from the recombinant E. coli K38(pGP1-2, pT7-4SH7.3) 60-fold, recovering 41.5% of dihydrolipoamide dehydrogenase activity. Microsequencing of the purified E3 component revealed an amino acid sequence which corresponded to the N-terminal amino acid sequence deduced from the nucleotide sequence of pdhL. The N-terminal region of PdhL comprising amino acids 1 to 112 was distinguished from all other known dihydrolipoamide dehydrogenases. It resembled the N terminus of dihydrolipoamide acyltransferases, and it contained one single lipoyl domain which was separated by an adjacent hinge region from the C-terminal region of the protein that exhibited high homology to classical dihydrolipoamide dehydrogenases.
The pyruvate dehydrogenase (PDH) complex of the gram-negative bacterium Zymomonas mobilis was purified to homogeneity. From 250 g of cells, we isolated 1 mg of PDH complex with a specific activity of 12.6 U/mg of protein. Analysis of subunit composition revealed a PDH (E1) consisting of the two subunits E1α (38 kDa) and E1β (56 kDa), a dihydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydrogenase (E3) of 50 kDa. The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, resembling the quaternary structures of PDH complexes from gram-positive bacteria and eukaryotes. The PDH complex-encoding genes were identified by hybridization experiments and sequence analysis in two separate gene regions in the genome of Z. mobilis. The genes pdhAα (1,065 bp) and pdhAβ (1,389 bp), encoding the E1α and E1β subunits of the E1 component, were located downstream of the gene encoding enolase. The pdhB (1,323 bp) and lpd (1,401 bp) genes, encoding the E2 and E3 components, were identified in an unrelated gene region together with a 450-bp open reading frame (ORF) of unknown function in the order pdhB-ORF2-lpd. Highest similarities of the gene products of the pdhAα, pdhAβ, and pdhB genes were found with the corresponding enzymes of Saccharomyces cerevisiae and other eukaryotes. Like the dihydrolipoamide acetyltransferases of S. cerevisiae and numerous other organisms, the product of the pdhB gene contains a single lipoyl domain. The E1β subunit PDH was found to contain an amino-terminal lipoyl domain, a property which is unique among PDHs.
We report the identification of novel inhibitors of Trypanosoma brucei 6PGDH enzyme by virtual fragment screening.
The enzyme 6-phosphogluconate dehydrogenase is a potential drug target for the parasitic protozoan Trypanosoma brucei, the causative organism of human African trypanosomiasis. This enzyme has a polar active site to accommodate the phosphate, hydroxyl and carboxylate groups of the substrate, 6-phosphogluconate. A virtual fragment screen was undertaken of the enzyme to discover starting points for the development of inhibitors which are likely to have appropriate physicochemical properties for an orally bioavailable compound. A virtual screening library was developed, consisting of compounds with functional groups that could mimic the phosphate group of the substrate, but which have a higher pKa. Following docking, hits were clustered and appropriate compounds purchased and assayed against the enzyme. Three fragments were identified that had IC50 values in the low micromolar range and good ligand efficiencies. Based on these initial hits, analogues were procured and further active compounds were identified. Some of the fragments identified represent potential starting points for a medicinal chemistry programme to develop potent drug-like inhibitors of the enzyme.
Virtual fragment screening; Trypanosoma brucei; 6-Phosphogluconate dehydrogenase
The pyruvate dehydrogenase multienzyme assembly (PDH) generates acetyl coenzyme A and reducing equivalents from pyruvate in a multiple-step process that is a nexus of central metabolism. We report crystal structures of the Geobacillus stearothermophilus PDH E1p subunit with ligands that mimic the prereaction complex and the postdecarboxylation product. The structures implicate residues that help to orient substrates, nurture intermediates, and organize surface loops so that they can engage a mobile lipoyl domain that receives the acetyl group and shuttles it to the next active site. The structural and enzymatic data suggest that H128β performs a dual role: first, as electrostatic catalyst of the reaction of pyruvate with the thiamine cofactor; and second, as a proton donor in the second reaction of acetyl group with the lipoate. We also identify I206α as a key residue in mediating the conformation of active-site loops. We propose that a simple conformational flip of the H271α side chain assists transfer of the acetyl group from thiamine cofactor to lipoyl domain in synchrony with reduction of the dithiolane ring.
present study focuses on fragmented deglycosylated pyranose
dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris. Fragmented deglycosylated PDH is formed from the deglycosylated
enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when
stored in a buffer solution at 4 °C. The remaining larger fragment
has a molecular weight of ∼46 kDa and exhibits higher volumetric
activity for glucose oxidation compared with the deglycosylated and
glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic
voltammetry were used to assess and compare the catalytic activity
of the three investigated forms of PDH, “wired” to graphite
electrodes with two different osmium redox polymers: [Os(4,4′-dimethyl-2,2′-bipyridine)2(poly(vinylimidazole))10Cl]+ [Os(dmbpy)PVI]
and [Os(4,4′-dimethoxy-2,2′-bipyridine)2(poly-(vinylimidazole))10Cl]+ [Os(dmobpy)PVI]. When “wired”
with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed
a pronounced increase in the current density with Jmax 13- and 6-fold higher than that observed for gPDH-
and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily
attractive for further biotechnological applications. An easier access
of the substrate to the active site and improved communication between
the enzyme and mediator matrix are suggested as the two main reasons
for the excellent performance of the fdgPDH when compared with that
of gPDH and dgPDH. Three of the four glycosites in PDH: N75, N175, and N252 were assigned using mass spectrometry
in conjunction with endoglycosidase treatment and tryptic digestion.
Determination of the asparagine residues carrying carbohydrate moieties
in PDH can serve as a solid background for production of recombinant
enzyme lacking glycosylation.
In trypanosomatids, glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentosephosphate pathway, is essential for the defense of the parasite against oxidative stress. Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana G6PDHs have been characterized. The parasites' G6PDHs contain a unique 37 amino acid long N-terminal extension that in T. cruzi seems to regulate the enzyme activity in a redox-state-dependent manner. T. brucei and T. cruzi G6PDHs, but not their Leishmania spp. counterpart, are inhibited, in an uncompetitive way, by steroids such as dehydroepiandrosterone and derivatives. The Trypanosoma enzymes are more susceptible to inhibition by these compounds than the human G6PDH. The steroids also effectively kill cultured trypanosomes but not Leishmania and are presently considered as promising leads for the development of new parasite-selective chemotherapeutic agents.
Dynamic models of metabolism can be useful in identifying potential drug targets, especially in unicellular organisms. A model of glycolysis in the causative agent of human African trypanosomiasis, Trypanosoma brucei, has already shown the utility of this approach. Here we add the pentose phosphate pathway (PPP) of T. brucei to the glycolytic model. The PPP is localized to both the cytosol and the glycosome and adding it to the glycolytic model without further adjustments leads to a draining of the essential bound-phosphate moiety within the glycosome. This phosphate “leak” must be resolved for the model to be a reasonable representation of parasite physiology. Two main types of theoretical solution to the problem could be identified: (i) including additional enzymatic reactions in the glycosome, or (ii) adding a mechanism to transfer bound phosphates between cytosol and glycosome. One example of the first type of solution would be the presence of a glycosomal ribokinase to regenerate ATP from ribose 5-phosphate and ADP. Experimental characterization of ribokinase in T. brucei showed that very low enzyme levels are sufficient for parasite survival, indicating that other mechanisms are required in controlling the phosphate leak. Examples of the second type would involve the presence of an ATP:ADP exchanger or recently described permeability pores in the glycosomal membrane, although the current absence of identified genes encoding such molecules impedes experimental testing by genetic manipulation. Confronted with this uncertainty, we present a modeling strategy that identifies robust predictions in the context of incomplete system characterization. We illustrate this strategy by exploring the mechanism underlying the essential function of one of the PPP enzymes, and validate it by confirming the model predictions experimentally.
Mathematical models have been valuable tools for investigating the complex behaviors of metabolism. Due to incomplete knowledge of biological systems, these models contain inevitable uncertainty. This uncertainty is present in the measured or estimated parameter values, but also in the structure of the metabolic network. In this paper we increase the coverage of a particularly well studied model of glucose metabolism in Trypanosoma brucei, a tropical parasite that causes African sleeping sickness, by extending it with an additional pathway in two compartments. During this modeling process we highlighted uncertainties in parameter values and network structure and used these to formulate new hypotheses which were subsequently tested experimentally. The models were improved with the experimentally derived data, but uncertainty remained concerning the exact topology of the system. These models allowed us to investigate the effects of the loss of one enzyme, 6-phosphogluconate dehydrogenase. By taking uncertainty into account, the models demonstrated that the loss of this enzyme is lethal to the parasite by a mechanism different than that in other organisms. Our methodology shows how formally introducing uncertainty into model building provides robust model behavior that is independent of the network structure or parameter values.
Methionyl-tRNA synthetase of Trypanosoma brucei (TbMetRS) is an important target in the development of new antitrypanosomal drugs. The enzyme is essential, highly flexible and displaying a large degree of changes in protein domains and binding pockets in the presence of substrate, product and inhibitors. Targeting this protein will benefit from a profound understanding of how its structure adapts to ligand binding. A series of urea-based inhibitors (UBIs) has been developed with IC50 values as low as 19 nM against the enzyme. The UBIs were shown to be orally available and permeable through the blood-brain barrier, and are therefore candidates for development of drugs for the treatment of late stage human African trypanosomiasis. Here, we expand the structural diversity of inhibitors from the previously reported collection and tested for their inhibitory effect on TbMetRS and on the growth of T. brucei cells. The binding modes and binding pockets of 14 UBIs are revealed by determination of their crystal structures in complex with TbMetRS at resolutions between 2.2 Å to 2.9 Å. The structures show binding of the UBIs through conformational selection, including occupancy of the enlarged methionine pocket and the auxiliary pocket. General principles underlying the affinity of UBIs for TbMetRS are derived from these structures, in particular the optimum way to fill the two binding pockets. The conserved auxiliary pocket might play a role in binding tRNA. In addition, a crystal structure of a ternary TbMetRS•inhibitor•AMPPCP complex indicates that the UBIs are not competing with ATP for binding, instead are interacting with ATP through hydrogen bond. This suggests a possibility that a general ‘ATP-engaging’ binding mode can be utilized for the design and development of inhibitors targeting tRNA synthetases of other disease-causing pathogen.
Infection by the protozoan parasite Trypanosoma brucei causes sleeping sickness, also called human African trypanosomiasis. Without treatment, the disease is fatal yet current therapeutic options are inadequate and better medicines are needed. We have previously reported several potent inhibitors of T. brucei methionyl-tRNA synthetase, an essential enzyme involved in the protein biosynthesis. Recently, a new series of the inhibitors was synthesized which has improved membrane permeability over the earlier inhibitors. When applied to mouse with T. brucei infection, the new compounds are orally available and reach the central nervous system to reduce parasite loads, and therefore are promising molecules to be developed into antitrypanosomal drug. Here, more inhibitors from this series are reported and tested for their activities. High resolution crystal structures were determined that revealed how these inhibitors bind to the target enzyme. The binding pockets of these inhibitors are thoroughly explored, providing profound insights which are beneficial for further development of MetRS inhibitors against sleeping sickness. A ternary complex of the enzyme, an inhibitor, and an ATP analogue was also determined, indicates that the inhibitor does not compete with ATP for binding. Based on this, a general approach to use inhibitors that engage ATP for binding to tRNA synthetases is proposed.
In the yeast Kluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH in K. lactis led us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+ bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activity in vivo. The appearance of a new G6PDH-specific activity band, following incubation of Saccharomyces cerevisiae and human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes.
The protein pirin, which is involved in a variety of biological processes, is conserved from prokaryotic microorganisms, fungi, and plants to mammals. It acts as a transcriptional cofactor or an apoptosis-related protein in mammals and is involved in seed germination and seedling development in plants. In prokaryotes, while pirin is stress induced in cyanobacteria and may act as a quercetinase in Escherichia coli, the functions of pirin orthologs remain mostly uncharacterized. We show that the Serratia marcescens pirin (pirinSm) gene encodes an ortholog of pirin protein. Protein pull-down and bacterial two-hybrid assays followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization-tandem mass spectrometry analyses showed the pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with the pirinSm gene. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity are inhibited by the pirinSm gene in S. marcescens CH-1. The S. marcescens CH-1 pirinSm gene was subsequently mutated by insertion-deletion homologous recombination. Accordingly, the PDH E1 and PDH enzyme complex activities and cellular ATP concentration increased up to 250%, 140%, and 220%, respectively, in the S. marcescens CH-1 pirinSm mutant. Concomitantly, the cellular NADH/NAD+ ratio increased in the pirinSm mutant, indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that the pirinSm gene plays a regulatory role in the process of pyruvate catabolism to acetyl coenzyme A through interaction with the PDH E1 subunit and inhibiting PDH enzyme complex activity in S. marcescens CH-1, and they suggest that pirinSm is an important protein involved in determining the direction of pyruvate metabolism towards either the TCA cycle or the fermentation pathways.
RNAi and enzymatic studies have shown the importance of 6-phosphogluconate dehydrogenase (6-PGDH) in Trypanosoma brucei for the parasite survival and make it an attractive drug target for the development of new treatments against human African trypanosomiasis. 2,3-O-Isopropylidene-4-erythrono hydroxamate is a potent inhibitor of parasite Trypanosoma brucei 6-phosphogluconate dehydrogenase (6-PGDH), the third enzyme of the pentose phosphate pathway. However, this compound does not have trypanocidal activity due to its poor membrane permeability. Consequently, we have previously reported a prodrug approach to improve the antiparasitic activity of this inhibitor by converting the phosphate group into a less charged phosphate prodrug. The activity of prodrugs appeared to be dependent on their stability in phosphate buffer. Here we have successfully further extended the development of the aryl phosphoramidate prodrugs of 2,3-O-isopropylidene-4-erythrono hydroxamate by synthesizing a small library of phosphoramidates and evaluating their biological activity and stability in a variety of assays. Some of the compounds showed high trypanocidal activity and good correlation of activity with their stability in fresh mouse blood.
Glycerol-3-phosphate dehydrogenases (G3PDHs) constitute a shuttle that serves for regeneration of NAD+ reduced during glycolysis. This NAD-dependent enzyme is employed in glycolysis and produces glycerol-3-phosphate from dihydroxyacetone phosphate, while its flavin adenine dinucleotide (FAD)-dependent homologue catalyzes a reverse reaction coupled to the respiratory chain. Trypanosoma brucei possesses two FAD-dependent G3PDHs. While one of them (mitochondrial G3PDH [mtG3PDH]) has been attributed to the mitochondrion and seems to be directly involved in G3PDH shuttle reactions, the function of the other enzyme (putative G3PDH [putG3PDH]) remains unknown. In this work, we used RNA interference and protein overexpression and tagging to shed light on the relative contributions of both FAD-G3PDHs to overall cellular metabolism. Our results indicate that mtG3PDH is essential for the bloodstream stage of T. brucei, while in the procyclic stage the enzyme is dispensable. Expressed putG3PDH-V5 was localized to the mitochondrion, and the data obtained by digitonin permeabilization, Western blot analysis, and immunofluorescence indicate that putG3PDH is located within the mitochondrion.
Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible for oxidation, whereas 2-fluorinated glucose performed poorly (C3 accessible), indicating that the glucose C2 position is the primary site of attack.
Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei.
Methods and Findings
We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002.
The mature domain of our TbCat•CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat•CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain•K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.
Proteases are ubiquitous in all forms of life and catalyze the enzymatic degradation of proteins. These enzymes regulate and coordinate a vast number of cellular processes and are therefore essential to many organisms. While serine proteases dominate in mammals, parasitic organisms commonly rely on cysteine proteases of the Clan CA family throughout their lifecycle. Clan CA cysteine proteases are therefore regarded as promising targets for the selective design of drugs to treat parasitic diseases, such as Human African Trypanosomiasis caused by Trypanosoma brucei. The genomes of kinetoplastids such as Trypanosoma spp. and Leishmania spp. encode two Clan CA C1 family cysteine proteases and in T. brucei these are represented by rhodesain and TbCatB. We have determined three-dimensional structures of these two enzymes as part of our ongoing efforts to synthesize more effective anti-trypanosomal drugs.
The Leishmania genus comprises up to 35 species, some with status still under discussion. The multilocus sequence typing (MLST)—extensively used for bacteria—has been proposed for pathogenic trypanosomatids. For Leishmania, however, a detailed analysis and revision on the taxonomy is still required. We have partially sequenced four housekeeping genes—glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), mannose phosphate isomerase (MPI) and isocitrate dehydrogenase (ICD)—from 96 Leishmania (Viannia) strains and assessed their discriminatory typing capacity. The fragments had different degrees of diversity, and are thus suitable to be used in combination for intra- and inter-specific inferences. Species-specific single nucleotide polymorphisms were detected, but not for all species; ambiguous sites indicating heterozygosis were observed, as well as the putative homozygous donor. A large number of haplotypes were detected for each marker; for 6PGD a possible ancestral allele for L. (Viannia) was found. Maximum parsimony-based haplotype networks were built. Strains of different species, as identified by multilocus enzyme electrophoresis (MLEE), formed separated clusters in each network, with exceptions. NeighborNet of concatenated sequences confirmed species-specific clusters, suggesting recombination occurring in L. braziliensis and L. guyanensis. Phylogenetic analysis indicates L. lainsoni and L. naiffi as the most divergent species and does not support L. shawi as a distinct species, placing it in the L. guyanensis cluster. BURST analysis resulted in six clonal complexes (CC), corresponding to distinct species. The L. braziliensis strains evaluated correspond to one widely geographically distributed CC and another restricted to one endemic area. This study demonstrates the value of systematic multilocus sequence analysis (MLSA) for determining intra- and inter-species relationships and presents an approach to validate the species status of some entities. Furthermore, it contributes to the phylogeny of L. (Viannia) and might be helpful for epidemiological and population genetics analysis based on haplotype/diplotype determinations and inferences.
Leishmania is a protozoan genus comprising many species, some associated with a human neglected disease called leishmaniasis. This parasite is found worldwide and is transmitted by sand flies, having numerous domestic and sylvatic animals as reservoirs. Leishmania is genetically and ecologically diverse and it has been argued that this has an impact on the epidemiology of the disease. Many typing methods have been proposed for the study of this diversity, although a generally agreed methodology is still required. Also, there is still a lack of consensus on the validity of some species. Multilocus sequence typing (MLST) is a method for studying the population structure and diversity of pathogens, but before an MLST scheme can be proposed it is essential to undertake a detailed analysis and selection of the sequences that are to be included in the system. Here, we sequenced four gene fragments of 96 L. (Viannia) strains, representing most species from this subgenus. Our results showed a good agreement between the current species assignment and the multilocus sequence analysis. Evidence of genetic recombination was found and the phylogenetic relationships were determined. Overall the results point to the feasibility of an MLST scheme for Leishmania and indicate that the four gene fragments analyzed could form part of this typing system. This will certainly be a valuable approach for taxonomy, population genetics, and epidemiological studies of this pathogen.
RNA editing ligase 1 (TbREL1) is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD) simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.
RNA editing ligase 1 (TbREL1) is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. Here we use molecular dynamics simulations to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed. During the apo simulations, a significant structural rearrangement of the enzyme's active site opens an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors against this protozoan parasite. State-of-the-art electrostatics calculations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.
The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas' disease chemotherapy is sterol 14α-demethylase (CYP51), a cytochrome P450 enzyme involved in biosynthesis of membrane sterols.
In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51Mt), we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51Mt. Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51Tc, demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine) of a single residue at a critical position in the active site.
CYP51Mt-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51Tc.
Enzyme sterol 14α-demethylase (CYP51) is a well-established target for anti-fungal therapy and is a prospective target for Chagas' disease therapy. We previously identified a chemical scaffold capable of delivering a variety of chemical structures into the CYP51 active site. In this work the binding modes of several second generation compounds carrying this scaffold were determined in high-resolution co-crystal structures with CYP51 of Mycobacterium tuberculosis. Subsequent assays against CYP51 in Trypanosoma cruzi, the agent of Chagas' disease, demonstrated that two of the compounds bound tightly to the enzyme. Both were tested for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. This compound is currently being evaluated in animal models of Chagas' disease. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability of a single amino acid residue at a critical position in the active site. Our work is aimed at rational design of potent and highly selective CYP51 inhibitors with potential to become therapeutic drugs. Drug selectivity to prevent host–pathogen cross-reactivity is pharmacologically important, because CYP51 is present in human host.
A previous study reported that the Tn5-induced poly(3-hydroxybutyric acid) (PHB)-leaky mutant Ralstonia eutropha H1482 showed a reduced PHB synthesis rate and significantly lower dihydrolipoamide dehydrogenase (DHLDH) activity than the wild-type R. eutropha H16 but similar growth behavior. Insertion of Tn5 was localized in the pdhL gene encoding the DHLDH (E3 component) of the pyruvate dehydrogenase complex (PDHC). Taking advantage of the available genome sequence of R. eutropha H16, observations were verified and further detailed analyses and experiments were done. In silico genome analysis revealed that R. eutropha possesses all five known types of 2-oxoacid multienzyme complexes and five DHLDH-coding genes. Of these DHLDHs, only PdhL harbors an amino-terminal lipoyl domain. Furthermore, insertion of Tn5 in pdhL of mutant H1482 disrupted the carboxy-terminal dimerization domain, thereby causing synthesis of a truncated PdhL lacking this essential region, obviously leading to an inactive enzyme. The defined ΔpdhL deletion mutant of R. eutropha exhibited the same phenotype as the Tn5 mutant H1482; this excludes polar effects as the cause of the phenotype of the Tn5 mutant H1482. However, insertion of Tn5 or deletion of pdhL decreases DHLDH activity, probably negatively affecting PDHC activity, causing the mutant phenotype. Moreover, complementation experiments showed that different plasmid-encoded E3 components of R. eutropha H16 or of other bacteria, like Burkholderia cepacia, were able to restore the wild-type phenotype at least partially. Interestingly, the E3 component of B. cepacia possesses an amino-terminal lipoyl domain, like the wild-type H16. A comparison of the proteomes of the wild-type H16 and of the mutant H1482 revealed striking differences and allowed us to reconstruct at least partially the impressive adaptations of R. eutropha H1482 to the loss of PdhL on the cellular level.
We have cloned the operon coding for the Bacillus subtilis S complex, which has been proposed to be a component in protein secretion machinery. A lambda gt10 library of B. subtilis was screened with antiserum directed against the Staphylococcus aureus membrane-bound ribosome protein complex, which is homologous to the B. subtilis S complex. Two positive overlapping lambda clones were sequenced. The S-complex operon, 5 kilobases in size, was shown to contain four open reading frames and three putative promoters, which are located upstream of the first, the third, and the last gene. The four proteins encoded by the operon are 42, 36, 48, and 50 kilodaltons in size. All of these proteins were recognized by antisera separately raised against each protein of the S. aureus membrane-bound ribosome protein and B. subtilis S complexes, thus verifying the S-complex identity of the lambda clones. Sequence analysis revealed that all four proteins of the B. subtilis S complex are homologous to the four subunits of the human pyruvate dehydrogenase (PDH). Also, the N terminus of the 48-kilodalton protein was found to have 70% amino acid identity with the N-terminal 211 amino acids, determined so far, from the E2 subunit of B. stearothermophilus PDH. Furthermore, chromosomal mapping of the S-complex operon gave a linkage to a marker gene located close to the previously mapped B. subtilis PDH genes. Thus, the S complex is evidently identical to the B. subtilis PDH, which has been shown to contain four subunits with molecular weights very similar to those of the S complex. Therefore, we propose that the S complex is not a primary component of protein secretion.
The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 Å resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the β6-α6 loop and α6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.
Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD+-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.
Trans-sialidase (TS), a virulence factor from Trypanosoma cruzi, is an enzyme playing key roles in the biology of this protozoan parasite. Absent from the mammalian host, it constitutes a potential target for the development of novel chemotherapeutic drugs, an urgent need to combat Chagas' disease. TS is involved in host cell invasion and parasite survival in the bloodstream. However, TS is also actively shed by the parasite to the bloodstream, inducing systemic effects readily detected during the acute phase of the disease, in particular, hematological alterations and triggering of immune cells apoptosis, until specific neutralizing antibodies are elicited. These antibodies constitute the only known submicromolar inhibitor of TS's catalytic activity. We now report the identification and detailed characterization of a neutralizing mouse monoclonal antibody (mAb 13G9), recognizing T. cruzi TS with high specificity and subnanomolar affinity. This mAb displays undetectable association with the T. cruzi superfamily of TS-like proteins or yet with the TS-related enzymes from Trypanosoma brucei or Trypanosoma rangeli. In immunofluorescence assays, mAb 13G9 labeled 100% of the parasites from the infective trypomastigote stage. This mAb also reduces parasite invasion of cultured cells and strongly inhibits parasite surface sialylation. The crystal structure of the mAb 13G9 antigen-binding fragment in complex with the globular region of T. cruzi TS was determined, revealing detailed molecular insights of the inhibition mechanism. Not occluding the enzyme's catalytic site, the antibody performs a subtle action by inhibiting the movement of an assisting tyrosine (Y119), whose mobility is known to play a key role in the trans-glycosidase mechanism. As an example of enzymatic inhibition involving non-catalytic residues that occupy sites distal from the substrate-binding pocket, this first near atomic characterization of a high affinity inhibitory molecule for TS provides a rational framework for novel strategies in the design of chemotherapeutic compounds.
Chagas' disease, or American trypanosomiasis, is an endemic illness that affects approximately 8 million people in Latin America. The etiologic agent is the protozoan parasite Trypanosoma cruzi. To survive in the mammalian host and invade its cells, leading to the chronic infection, the parasite incorporates a charged carbohydrate (sialic acid). However, the parasite is unable to synthesize sialic acid, having to scavenge it from the host's sialo-glycoconjugates, through a transglycosylation reaction catalyzed by the enzyme trans-sialidase, which is unique to these organisms. We have obtained a monoclonal antibody that fully inhibits T. cruzi trans-sialidase actually being, at the best of our knowledge, the most potent inhibitor available. We now report a complete characterization of this neutralizing monoclonal antibody, at the functional and molecular levels. The antibody displays very high affinity and specificity for the T. cruzi enzyme, labels the parasites' surface and effectively blocks its sialylation and host cell invasion capacities. The determination of the 3D structure of the enzyme-antibody immunocomplex by X ray diffraction, allowed us to unveil the inhibition mechanism, providing clues for rational drug design. Given that sialidases are virulence factors in several pathogenic microorganisms, the reported data shall help to expand informative knowledge in this area.
The pdhABCD operon of Bacillus subtilis encodes the pyruvate decarboxylase (E1α and E1β), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH). There are two promoters: one for the entire operon and an internal one in front of the pdhC gene. The latter may serve to ensure adequate quantities of the E2 and E3 subunits, which are needed in greater amounts than E1α and E1β. Disruptions of the pdhB, pdhC, and pdhD genes were isolated, but attempts to construct a pdhA mutant were unsuccessful, suggesting that E1α is essential. The three mutants lacked PDH activity, were unable to grow on glucose and grew poorly in an enriched medium. The pdhB and pdhC mutants sporulated to only 5% of the wild-type level, whereas the pdhD mutant strain sporulated to 55% of the wild-type level. This difference indicated that the sporulation defect of the pdhB and pdhC mutant strains was due to a function(s) of these subunits independent of enzymatic activity. Growth, but not low sporulation, was enhanced by the addition of acetate, glutamate, succinate, and divalent cations. Results from the expression of various spo-lacZ fusions revealed that the pdhB mutant was defective in the late stages of engulfment or membrane fusion (stage II), whereas the pdhC mutant was blocked after the completion of engulfment (stage III). This analysis was confirmed by fluorescent membrane staining. The E1β and E2 subunits which are present in the soluble fraction of sporulating cells appear to function independently of enzymatic activity as checkpoints for stage II-III of sporulation.