The gene encoding the putative mevalonate diphosphate decarboxylase, an enzyme from the mevalonate pathway of isoprenoid precursor biosynthesis, has been cloned from T. brucei. Recombinant protein has been expressed, purified and highly ordered crystals obtained and characterized to aid the structure–function analysis of this enzyme.
Mevalonate diphosphate decarboxylase catalyses the last and least well characterized step in the mevalonate pathway for the biosynthesis of isopentenyl pyrophosphate, an isoprenoid precursor. A gene predicted to encode the enzyme from Trypanosoma brucei has been cloned, a highly efficient expression system established and a purification protocol determined. The enzyme gives monoclinic crystals in space group P21, with unit-cell parameters a = 51.5, b = 168.7, c = 54.9 Å, β = 118.8°. A Matthews coefficient V
M of 2.5 Å3 Da−1 corresponds to two monomers, each approximately 42 kDa (385 residues), in the asymmetric unit with 50% solvent content. These crystals are well ordered and data to high resolution have been recorded using synchrotron radiation.
decarboxylases; mevalonate biosynthesis; isoprenoids; Trypanosoma
Two, simple, C5 compounds, dimethylally diphosphate and isopentenyl diphosphate, are the universal precursors of isoprenoids, a large family of natural products involved in numerous important biological processes. Two distinct biosynthetic pathways have evolved to supply these precursors. Humans use the mevalonate route whilst many species of bacteria including important pathogens, plant chloroplasts and apicomplexan parasites exploit the non-mevalonate pathway. The absence from humans, combined with genetic and chemical validation suggests that the non-mevalonate pathway holds the potential to support new drug discovery programmes targeting Gram-negative bacteria and the apicomplexan parasites responsible for causing serious human diseases, and also infections of veterinary importance. The non-mevalonate pathway relies on eight enzyme-catalyzed stages exploiting a range of cofactors and metal ions. A wealth of structural and mechanistic data, mainly derived from studies of bacterial enzymes, now exists for most components of the pathway and these will be described. Particular attention will be paid to how these data inform on the apicomplexan orthologues concentrating on the enzymes from Plasmodium spp.; these cause malaria, one the most important parasitic diseases in the world today.
Antimicrobial drug discovery; isoprenoid precursor biosynthesis; malaria; structure-based inhibitor discovery; toxoplasmosis
The mevalonate pathway is utilized for the biosynthesis of isoprenoids in many bacterial, eukaryotic, and archaeal organisms. Based on previous reports of its feedback inhibition, mevalonate kinase (MVK) may play an important regulatory role in the biosynthesis of mevalonate pathway-derived compounds. Here we report the purification, kinetic characterization, and inhibition analysis of the MVK from the archaeon Methanosarcina mazei. The inhibition of the M. mazei MVK by the following metabolites derived from the mevalonate pathway was explored: dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isopentenyl monophosphate (IP), and diphosphomevalonate. M. mazei MVK was not inhibited by DMAPP, GPP, FPP, diphosphomevalonate, or IP, a proposed intermediate in an alternative isoprenoid pathway present in archaea. Our findings suggest that the M. mazei MVK represents a distinct class of mevalonate kinases that can be differentiated from previously characterized MVKs based on its inhibition profile.
4-Diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE) catalyses the ATP-dependent conversion of 4-diphosphocytidyl-2C-methyl-d-erythritol (CDPME) to 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate with the release of ADP. This reaction occurs in the non-mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti-infective drugs. We set out to characterize the biochemical properties, determinants of molecular recognition and reactivity of IspE and report the cloning and purification of recombinant Aquifex aeolicus IspE (AaIspE), kinetic data, metal ion, temperature and pH dependence, crystallization and structure determination of the enzyme in complex with CDP, CDPME and ADP. In addition, 4-fluoro-3,5-dihydroxy-4-methylpent-1-enylphosphonic acid (compound 1) was designed to mimic a fragment of the substrate, a synthetic route to 1 was elucidated and the complex structure determined. Surprisingly, this ligand occupies the binding site for the ATP α-phosphate not the binding site for the methyl-d-erythritol moiety of CDPME. Gel filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution. The enzyme displays the characteristic α/β galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains. Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism. The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents.
enzyme–ligand complex; GHMP kinase; isoprenoid biosynthesis; molecular recognition; non-mevalonate pathway
The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)–pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.
Glycolysis and glyconeogenesis play crucial roles in the ATP supply and synthesis of glycoconjugates, important for the viability and virulence, respectively, of the human-pathogenic stages of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. These pathways are, therefore, candidate targets for antiparasite drugs. The glycolytic/gluconeogenic enzyme enolase is generally highly conserved, with similar overall fold and identical catalytic residues in all organisms. Nonetheless, potentially important differences exist between the trypanosomatid and host enzymes, with three unique, reactive residues close to the active site of the former that might be exploited for the development of new drugs. In addition, enolase is found both in the secretome and in association with the surface of Leishmania spp. where it probably functions as plasminogen receptor, playing a role in the parasite's invasiveness and virulence, a function possibly also present in the other trypanosomatids. This location and possible function of enolase offer additional perspectives for both drug discovery and vaccination.
It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent Km values for R,S-mevalonate and ATP were 41 and 339 μM, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the Ki values were 46 and 45 μM for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (Ki difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.
The biosynthesis of isopentenyl diphosphate (IPP) from either the mevalonate (MVA) or the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway provides the key metabolite for primary and secondary isoprenoid biosynthesis. Isoprenoid metabolism plays crucial roles in membrane stability, steroid biosynthesis, vitamin production, protein localization, defense and communication, photoprotection, sugar transport, and glycoprotein biosynthesis. Recently, an alternative branch of the MVA pathway was discovered in the archaeon Methanocaldococcus jannaschii involving a small molecule kinase, isopentenyl phosphate kinase (IPK). IPK belongs to the amino acid kinase (AAK) superfamily. In vitro, IPK phosphorylates isopentenyl monophosphate (IP) in an ATP and Mg2+-dependent reaction producing IPP. Here, we describe crystal structures of IPK from M. jannaschii refined to nominal resolutions of 2.0−2.8 Å. Notably, an active site histidine residue (His60) forms a hydrogen bond with the terminal phosphate of both substrate and product. This His residue serves as a marker for a subset of the AAK family that catalyzes phosphorylation of phosphate or phosphonate functional groups; the larger family includes carboxyl-directed kinases, which lack this active site residue. Using steady-state kinetic analysis of H60A, H60N, and H60Q mutants, the protonated form of the Nε2 nitrogen of His60 was shown to be essential for catalysis, most likely through hydrogen bond stabilization of the transition state accompanying transphosphorylation. Moreover, the structures served as the starting point for the engineering of IPK mutants capable of the chemoenzymatic synthesis of longer chain isoprenoid diphosphates from monophosphate precursors.
Mevalonate Kinase Deficiency (MKD) is a rare autosomal recessive inborn disorder of cholesterol biosynthesis caused by mutations in the mevalonate kinase (MK) gene, leading to MK enzyme decreased activity. The consequent shortage of mevalonate-derived isoprenoid compounds results in an inflammatory phenotype, caused by the activation of the NALP3 inflammasome that determines an increased caspase-1 activation and IL-1β release. In MKD, febrile temperature can further decrease the residual MK activity, leading to mevalonate pathway modulation and to possible disease worsening. We previously demonstrated that the administration of exogenous isoprenoids such as geraniol or the modulation of the enzymatic pathway with drugs, such as Tipifarnib, partially rescues the inflammatory phenotype associated with the defective mevalonic pathway. However, it has not been investigated yet how temperature can affect the success of these treatments. Thus, we investigated the effect of temperature on primary human monocytes from MKD patients. Furthermore the ability of geraniol and Tipifarnib to reduce the abnormal inflammatory response, already described at physiological temperature in MKD, was studied in a febrile condition. We evidenced the role of temperature in the modulation of the inflammatory events and suggested strongly considering this variable in future researches aimed at finding a treatment for MKD.
3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase is a critical enzyme in the mevalonate pathway that regulates the biosynthesis of cholesterol as well as isoprenoids that mediate the membrane association of certain GTPases. Blockade of this enzyme by atorvastatin (AT) inhibits the destructive proinflammatory T helper cell (Th)1 response during experimental autoimmune encephalomyelitis and may be beneficial in the treatment of multiple sclerosis and other Th1-mediated autoimmune diseases. Here we present evidence linking specific isoprenoid intermediates of the mevalonate pathway to signaling pathways that regulate T cell autoimmunity. We demonstrate that the isoprenoid geranylgeranyl-pyrophosphate (GGPP) mediates proliferation, whereas both GGPP and its precursor, farnesyl-PP, regulate the Th1 differentiation of myelin-reactive T cells. Depletion of these isoprenoid intermediates in vivo via oral AT administration hindered these T cell responses by decreasing geranylgeranylated RhoA and farnesylated Ras at the plasma membrane. This was associated with reduced extracellular signal–regulated kinase (ERK) and p38 phosphorylation and DNA binding of their cotarget c-fos in response to T cell receptor activation. Inhibition of ERK and p38 mimicked the effects of AT and induced a Th2 cytokine shift. Thus, by connecting isoprenoid availability to regulation of Th1/Th2 fate, we have elucidated a mechanism by which AT may suppress Th1-mediated central nervous system autoimmune disease.
Phosphomevalonate kinase (PMK) catalyzes the cation dependent reaction of mevalonate 5-phosphate with ATP to form mevalonate 5-diphosphate and ADP, a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis. Animal PMK proteins belong to the nucleoside monophosphate (NMP) kinase family. For many NMP kinases, multiple basic residues contribute to the neutralization of the negatively charged pentacoordinate phosphate reaction intermediate. Loss of basicity can result in catalytically impaired enzymes. Based on this precedent, conserved basic residues of human PMK have been mutated and purified forms of the mutated proteins have been kinetically and biophysically characterized. K48M and R73M mutants exhibit diminished Vmax values in both reaction directions (>1000-fold) with only slight Km perturbations (<10-fold). In both forward and reverse reactions, R110M exhibits a large (>10,000-fold) specific activity diminution. R111M exhibits substantially inflated Km values for mevalonate 5-phosphate and mevalonate 5-diphosphate (60 and 30-fold, respectively) as well as decreases (50-fold (forward) and 85-fold (reverse)) in Vmax. R84M also exhibits inflated Km values (50 and 33-fold for mevalonate 5-phosphate and mevalonate 5-diphosphate, respectively). The Ki values for R111M and R84M product inhibition by mevalonate 5-diphosphate are inflated by 45- and 63-fold; effects are comparable to the 30- and 38-fold inflations in Km for mevalonate 5-diphosphate. R141M exhibits little perturbation in Vmax (14-fold (forward) and 10-fold (reverse)) but has inflated Km values for ATP and ADP (48 and 136-fold, respectively). The Kd of ATP for R141M, determined by changes in tryptophan fluorescence, is inflated 27-fold compared to wt PMK. These data suggest that R110 is important to PMK catalysis, which is also influenced by K48 and R73. R111 and R84 contribute to binding of mevalonate 5-phosphate and R141 to binding of ATP.
Isoprenoids are a class of ubiquitous organic molecules synthesized from the five-carbon starter unit isopentenyl pyrophosphate (IPP). Comprising more than 30,000 known natural products, isoprenoids serve various important biological functions in many organisms. In bacteria, undecaprenyl pyrophosphate is absolutely required for the formation of cell wall peptidoglycan and other cell surface structures, while ubiquinones and menaquinones, both containing an essential prenyl moiety, are key electron carriers in respiratory energy generation. There is scant knowledge on the nature and regulation of bacterial isoprenoid pathways. In order to explore the cellular responses to perturbations in the mevalonate pathway, responsible for producing the isoprenoid precursor IPP in many gram-positive bacteria and eukaryotes, we constructed three strains of Staphylococcus aureus in which each of the mevalonate pathway genes is regulated by an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter. We used DNA microarrays to profile the transcriptional effects of downregulating the components of the mevalonate pathway in S. aureus and demonstrate that decreased expression of the mevalonate pathway leads to widespread downregulation of primary metabolism genes, an upregulation in virulence factors and cell wall biosynthetic determinants, and surprisingly little compensatory expression in other isoprenoid biosynthetic genes. We subsequently correlate these transcriptional changes with downstream metabolic consequences.
Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-d-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP.
biosynthesis; inhibitor; isoprenoid; MEP pathway; mevalonate pathway; terpenoid
In mycobacteria, the biosynthesis of the precursors to the essential isoprenoids, isopentenyl diphosphate and dimethylallyl pyrophosphate is carried out by the methylerythritol phosphate (MEP) pathway. This route of synthesis is absent in humans, who utilize the alternative mevalonate acid (MVA) route, thus making the enzymes of the MEP pathway of chemotherapeutic interest. One such identified target is the second enzyme of the pathway, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). Only limited information is currently available concerning the catalytic mechanism and structural dynamics of this enzyme, and only recently has a crystal structure of Mycobacterium tuberculosis species of this enzyme been resolved including all factors required for binding. Here, the dynamics of the enzyme is studied in complex with NADPH, Mn2+, in the presence and absence of the fosmidomycin inhibitor using conventional molecular dynamics and an enhanced sampling technique, Reversible Digitally Filtered Molecular Dynamics. The simulations reveal significant differences in the conformational dynamics of the vital catalytic loop between the inhibitor-free and inhibitor-bound enzyme complexes and highlight the contributions of conserved residues in this region. The substantial fluctuations observed suggest that DXR may be a promising target for computer-aided drug discovery through the relaxed complex method.
In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.
The role of peroxisomes in isoprenoid metabolism, especially in plants, has been questioned in several reports. A recent study of Sapir-Mir et al.1 revealed that the two isoforms of isopentenyl diphosphate (IPP) isomerase, catalyzing the isomerisation of IPP to dimethylallyl diphosphate (DMAPP) are found in the peroxisome. In this addendum, we provide additional data describing the peroxisomal localization of 5-phosphomevalonate kinase and mevalonate 5-diphosphate decarboxylase, the last two enzymes of the mevalonic acid pathway leading to IPP.2 This finding was reinforced in our latest report showing that a short isoform of farnesyl diphosphate, using IPP and DMAPP as substrates, is also targeted to the organelle.3 Therefore, the classical sequestration of isoprenoid biosynthesis between plastids and cytosol/ER can be revisited by including the peroxisome as an additional isoprenoid biosynthetic compartment within plant cells.
5-phosphomevalonate kinase; Arabidopsis thaliana; Catharanthus roseus; farnesyl diphosphate synthase; isoprenoid; mevalonate 5-diphosphate decarboxylase; mevalonic acid pathway; peroxisome
The activity of nerolidol, a sesquiterpene used as a food-flavoring agent and currently under testing as a skin penetration enhancer for the transdermal delivery of therapeutic drugs, was evaluated against Leishmania species. Nerolidol inhibited the growth of Leishmania amazonensis, L. braziliensis, and L. chagasi promastigotes and L. amazonensis amastigotes with in vitro 50% inhibitory concentrations of 85, 74, 75, and 67 μM, respectively. The treatment of L. amazonensis-infected macrophages with 100 μM nerolidol resulted in 95% reduction in infection rates. Inhibition of isoprenoid biosynthesis, as shown by reduced incorporation of [2-14C]mevalonic acid (MVA) or [1-14C]acetic acid precursors into dolichol, ergosterol, and ubiquinone, was observed in nerolidol-treated promastigotes. This drug effect can be attributed to the blockage of an early step in the mevalonate pathway, since incorporation of the precursor [1(n)-3H]farnesyl pyrophosphate in polyisoprenoids is not inhibited by nerolidol. L. amazonensis-infected BALB/c mice were treated with intraperitoneal doses of 100 mg/kg/day for 12 days or topically with 5 or 10% ointments for 4 weeks. Significant reduction of lesion sizes in nerolidol treated mice was observed for both treatment routes. However, long-term follow up indicated that the disease was not cured in this highly susceptible animal model. Nonetheless, the in vitro activity of nerolidol against these parasites may prove a useful tool for the development of new drugs for the treatment of leishmaniasis. In addition, biosynthesis of dolichols with 11 and 12 isoprene units was identified in Leishmania, as described for other trypanosomatids and Apicomplexa.
Mevalonate kinase deficiency (MKD) is a rare hereditary auto-inflammatory syndrome due to mutations in mevalonate kinase, the second enzyme of mevalonate pathway of cholesterol, and nonsterol-isoprenoids biosynthesis. The shortage of mevalonate-derived intermediates, and in particular of geranylgeranyl pyrophosphate (GGPP), has been linked with the activation of caspase-1 and thereby with the production of IL-1β, but the true concatenation of these two events has not been clarified yet. We hypothesized that inflammasomes could mediate the activation of caspase-1 due to the shortage of GGPP. We monitored the expression of the principal proteins (NALP1, NALP3 and IPAF) of the three known inflammasomes, first in a cellular model of MKD and then in two MKD patients, after bacterial lipopolysaccharide (LPS) stimulation. In healthy subjects, alendronate alone induced the expression of NALP1 and NALP3, and then together with LPS it induced a dramatic increase in NALP3 expression. In MKD patients, NALP3 expression was higher than in untreated healthy controls. Our results, although preliminary, showed that the inhibition of the mevalonate pathway led to a hyper-expression of NALP3, suggesting a possible involvement of NALP3-inflammasome in the activation of caspase-1 consequent to GGPP decrement. This is the first time that the involvement of the inflammasome complexes was shown in MKD pathogenesis.
mevalonate kinase deficiency; inflammasome; NALP3; caspase-1
Isoprenoid compounds are ubiquitous in nature, participating in important biological phenomena such as signal transduction, aerobic cellular respiration, photosynthesis, insect communication, and many others. They are derived from the 5-carbon isoprenoid substrates isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In Archaea and Eukarya, these building blocks are synthesized via the mevalonate pathway. However, the genes required to convert mevalonate phosphate (MP) to IPP are missing in several species of Archaea. An enzyme with isopentenyl phosphate kinase (IPK) activity was recently discovered in Methanocaldococcus jannaschii (MJ), suggesting a departure from the classical sequence of converting MP to IPP. We have determined the high-resolution crystal structures of isopentenyl phosphate kinases in complex with both substrates and products from Thermoplasma acidophilum (THA), as well as the IPK from Methanothermobacter thermautotrophicus (MTH), by means of single-wavelength anomalous diffraction (SAD) and molecular replacement. A histidine residue (His50) in THA IPK makes a hydrogen bond with the terminal phosphates of IP and IPP, poising these molecules for phosphoryl transfer through an in-line geometry. Moreover, a lysine residue (Lys14) makes hydrogen bonds with non-bridging oxygen atoms at Pα and Pγ and with the Pβ- Pγ bridging oxygen atom in ATP. These interactions suggest a transition state-stabilizing role for this residue. Lys14 is a part of a newly discovered “lysine triangle” catalytic motif in IPK’s that also includes Lys5 and Lys205. Moreover, His50, Lys5, Lys14, and Lys205 are conserved in all IPK’s and can therefore serve as fingerprints for identifying new homologues.
The glyoxalase system, comprising the metalloenzymes glyoxalase I (GLO1) and glyoxalase II (GLO2), is an almost universal metabolic pathway involved in the detoxification of the glycolytic byproduct methylglyoxal to d-lactate. In contrast to the situation with the trypanosomatid parasites Leishmania major and Trypanosoma cruzi, this trypanothione-dependent pathway is less well understood in the African trypanosome, Trypanosoma brucei. Although this organism possesses a functional GLO2, no apparent GLO1 gene could be identified in the T. brucei genome. The absence of GLO1 in T. brucei was confirmed by the lack of GLO1 activity in whole cell extracts, failure to detect a GLO1-like protein on immunoblots of cell lysates, and lack of d-lactate formation from methylglyoxal as compared to L. major and T. cruzi. T. brucei procyclics were found to be 2.4-fold and 5.7-fold more sensitive to methylglyoxal toxicity than T. cruzi and L. major, respectively. T. brucei also proved to be the least adept of the ‘Tritryp’ parasites in metabolizing methylglyoxal, producing l-lactate rather than d-lactate. Restoration of a functional glyoxalase system by expression of T. cruzi GLO1 in T. brucei resulted in increased resistance to methylglyoxal and increased conversion of methylglyoxal to d-lactate, demonstrating that GLO2 is functional in vivo. Procyclic forms of T. brucei possess NADPH-dependent methylglyoxal reductase and NAD+-dependent l-lactaldehyde dehydrogenase activities sufficient to account for all of the methylglyoxal metabolized by these cells. We propose that the predominant mechanism for methylglyoxal detoxification in the African trypanosome is via the methylglyoxal reductase pathway to l-lactate.
glyoxalase; lactate; methylglyoxal metabolism; Trypanosoma brucei; trypanothione
The structure of a triclinic crystal form of 4-diphosphocytidyl-2C-methyl-d-erythritol kinase has been determined. Comparisons with a previously reported monoclinic crystal form raise questions about our knowledge of the quaternary structure of this enzyme.
4-Diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE; EC 126.96.36.199) contributes to the 1-deoxy-d-xylulose 5-phosphate or mevalonate-independent biosynthetic pathway that produces the isomers isopentenyl diphosphate and dimethylallyl diphosphate. These five-carbon compounds are the fundamental building blocks for the biosynthesis of isoprenoids. The mevalonate-independent pathway does not occur in humans, but is present and has been shown to be essential in many dangerous pathogens, i.e. Plasmodium species, which cause malaria, and Gram-negative bacteria. Thus, the enzymes involved in this pathway have attracted attention as potential drug targets. IspE produces 4-diphosphosphocytidyl-2C-methyl-d-erythritol 2-phosphate by ATP-dependent phosphorylation of 4-diphosphocytidyl-2C-methyl-d-erythritol. A triclinic crystal structure of the Escherichia coli IspE–ADP complex with two molecules in the asymmetric unit was determined at 2 Å resolution and compared with a monoclinic crystal form of a ternary complex of E. coli IspE also with two molecules in the asymmetric unit. The molecular packing is different in the two forms. In the asymmetric unit of the triclinic crystal form the substrate-binding sites of IspE are occluded by structural elements of the partner, suggesting that the ‘triclinic dimer’ is an artefact of the crystal lattice. The surface area of interaction in the triclinic form is almost double that observed in the monoclinic form, implying that the dimeric assembly in the monoclinic form may also be an artifact of crystallization.
mevalonate-independent pathway; isoprenoid biosynthesis; kinases
The cell surface glycoconjugates of trypanosomatid parasites are intimately involved in parasite survival, infectivity, and virulence in their insect vectors and mammalian hosts. Although there is a considerable body of work describing their structure, biosynthesis, and function, little is known about the sugar nucleotide pools that fuel their biosynthesis. In order to identify and quantify parasite sugar nucleotides, we developed an analytical method based on liquid chromatography-electrospray ionization-tandem mass spectrometry using multiple reaction monitoring. This method was applied to the bloodstream and procyclic forms of Trypanosoma brucei, the epimastigote form of T. cruzi, and the promastigote form of Leishmania major. Five sugar nucleotides, GDP-α-d-mannose, UDP-α-d-N-acetylglucosamine, UDP-α-d-glucose, UDP-α-galactopyranose, and GDP-β-l-fucose, were common to all three species; one, UDP-α-d-galactofuranose, was common to T. cruzi and L. major; three, UDP-β-l-rhamnopyranose, UDP-α-d-xylose, and UDP-α-d-glucuronic acid, were found only in T. cruzi; and one, GDP-α-d-arabinopyranose, was found only in L. major. The estimated demands for each monosaccharide suggest that sugar nucleotide pools are turned over at very different rates, from seconds to hours. The sugar nucleotide survey, together with a review of the literature, was used to define the routes to these important metabolites and to annotate relevant genes in the trypanosomatid genomes.
Isoprenoids constitute an important class of biomolecules that participate in many different cellular processes. Most available detection methods only allow the identification of one or two specific non-sterol isoprenoid intermediates following radioactive or fluorescent labeling. We here report a rapid, non-radioactive and sensitive procedure for the simultaneous detection and quantification of the 8 main non-sterol intermediates of the isoprenoid biosynthesis pathway by means of tandem mass spectrometry. Intermediates were analyzed by HPLC-MS/MS in the multiple reaction monitoring mode using a silica-based C18 HPLC column. For quantification, their stable-isotope-labeled analogues were used as internal standards. HepG2 cells were used to validate the method. Mevalonate, phosphomevalonate and the 6 subsequent isoprenoid-pyrophosphates were readily determined with detection limits ranging from 0.03 to 1.0 μmol/L. The intra- and interassay variations for HepG2 cell homogenates supplemented with isoprenoid intermediates were 3.6–10.9% and 4.4–11.9%, respectively. Under normal culturing conditions, isoprenoid intermediates in HepG2 cells were below detection limits. However, incubation of the cells with pamidronate, an inhibitor of farnesyl pyrophosphate synthase, resulted in increased levels of MVA, IPP/DMAPP and GPP. This method will be suitable to measure profiles of isoprenoid intermediates in cells with compromised isoprenoid biosynthesis, and to determine the specificity of potential inhibitors of the pathway.
Isoprenoid biosynthesis; Mevalonate kinase deficiency; Mass spectrometry; Farnesyl pyrophosphate; Geranylgeranyl pyrophosphate
The glycosomes of trypanosomatids are essential organelles that are evolutionarily related to peroxisomes of other eukaryotes. The peroxisomal RING proteins - PEX2, PEX10 and PEX12 - comprise a network of integral membrane proteins that function in the matrix protein import cycle. Here, we describe PEX10 and PEX12 in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi. We expressed GFP fusions of each T. brucei coding region in procyclic form T. brucei, where they localized to glycosomes and behaved as integral membrane proteins. Despite the weak transmembrane predictions for TbPEX12, protease protection assays demonstrated that both the N and C termini are cytosolic, similar to mammalian PEX12. GFP fusions of T. cruzi PEX10 and L. major PEX12 also localized to glycosomes in T. brucei indicating that glycosomal membrane protein targeting is conserved across trypanosomatids.
Trypanosoma brucei; Trypanosoma cruzi; Leishmania major; Kinetoplastida; peroxisomes; glycosomes; RING; peroxins; PEX10; PEX12
The trypanosomatids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi cause some of the most debilitating diseases of humankind: cutaneous leishmaniasis, African sleeping sickness, and Chagas disease. These protozoa possess complex life cycles that involve development in mammalian and insect hosts, and a tightly coordinated cell cycle ensures propagation of the highly polarized cells. However, the ways in which the parasites respond to their environment and coordinate intracellular processes are poorly understood. As a part of an effort to understand parasite signaling functions, we report the results of a genome-wide analysis of protein kinases (PKs) of these three trypanosomatids.
Bioinformatic searches of the trypanosomatid genomes for eukaryotic PKs (ePKs) and atypical PKs (aPKs) revealed a total of 176 PKs in T. brucei, 190 in T. cruzi and 199 in L. major, most of which are orthologous across the three species. This is approximately 30% of the number in the human host and double that of the malaria parasite, Plasmodium falciparum. The representation of various groups of ePKs differs significantly as compared to humans: trypanosomatids lack receptor-linked tyrosine and tyrosine kinase-like kinases, although they do possess dual-specificity kinases. A relative expansion of the CMGC, STE and NEK groups has occurred. A large number of unique ePKs show no strong affinity to any known group. The trypanosomatids possess few ePKs with predicted transmembrane domains, suggesting that receptor ePKs are rare. Accessory Pfam domains, which are frequently present in human ePKs, are uncommon in trypanosomatid ePKs.
Trypanosomatids possess a large set of PKs, comprising approximately 2% of each genome, suggesting a key role for phosphorylation in parasite biology. Whilst it was possible to place most of the trypanosomatid ePKs into the seven established groups using bioinformatic analyses, it has not been possible to ascribe function based solely on sequence similarity. Hence the connection of stimuli to protein phosphorylation networks remains enigmatic. The presence of numerous PKs with significant sequence similarity to known drug targets, as well as a large number of unusual kinases that might represent novel targets, strongly argue for functional analysis of these molecules.