Type I and type II fatty acid biosynthesis in Eimeria tenella: enoyl reductase activity and structure.

Apicomplexan parasites of the genus Eimeria are the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. Recent results show that Eimeria tenella harbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. In related parasites, enoyl reductase is one component of a type II fatty acid synthase (FAS) and has proven to be an attractive target for antimicrobial compounds. We cloned and expressed the mature form of E. tenella enoyl reductase (EtENR) for biochemical and structural studies. Recombinant EtENR exhibits NADH-dependent enoyl reductase activity and is inhibited by triclosan with an IC50 value of 60 nm. The crystal structure of EtENR reveals overall similarity with other ENR enzymes; however, the active site of EtENR is unoccupied, a state rarely observed in other ENR structures. Furthermore, the position of the central beta-sheet appears to block NADH binding and would require significant movement to allow NADH binding, a feature not previously seen in the ENR family. We analysed the E. tenella genomic database for orthologues of well-characterized bacterial and apicomplexan FAS enzymes and identified 6 additional genes, suggesting that E. tenella contains a type II FAS capable of synthesizing saturated, but not unsaturated, fatty acids. Interestingly, we also identified sequences that appear to encode multifunctional type I FAS enzymes, a feature also observed in Toxoplasma gondii, highlighting the similarity between these apicomplexan parasites.

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

Delivery of antimicrobials into parasites.

To eliminate apicomplexan parasites, inhibitory compounds must cross host cell, parasitophorous vacuole, and parasite membranes and cyst walls, making delivery challenging. Here, we show that short oligomers of arginine enter Toxoplasma gondii tachyzoites and encysted bradyzoites. Triclosan, which inhibits enoyl-ACP reductase (ENR), conjugated to arginine oligomers enters extracellular tachyzoites, host cells, tachyzoites inside parasitophorous vacuoles within host cells, extracellular bradyzoites, and bradyzoites within cysts. We identify, clone, and sequence T. gondii enr and produce and characterize enzymatically active, recombinant ENR. This enzyme has the requisite amino acids to bind triclosan. Triclosan released after conjugation to octaarginine via a readily hydrolyzable ester linkage inhibits ENR activity, tachyzoites in vitro, and tachyzoites in mice. Delivery of an inhibitor to a microorganism via conjugation to octaarginine provides an approach to transporting antimicrobials and other small molecules to sequestered parasites, a model system to characterize transport across multiple membrane barriers and structures, a widely applicable paradigm for treatment of active and encysted apicomplexan and other infections, and a generic proof of principle for a mechanism of medicine delivery.

Influence of human p16(INK4) and p21(CIP1) on the in vitro activity of recombinant Plasmodium falciparum cyclin-dependent protein kinases.

The regulatory mechanisms of most cyclin dependent protein kinases (CDKs) are well understood and are highly conserved in eukaryotes. CDKs from the malaria parasite, Plasmodium falciparum, appear to be regulated in a similar manner with regard to cyclin binding and phosphorylation. In order to further understand their regulatory mechanisms, we examined two classes of cyclin dependent kinase inhibitors (CDIs) to inhibit a panel of plasmodial CDKs. We find that Pfmrk and PfPK5 are inhibited by heterologous p21(CIP1) with varying degrees of inhibition. In contrast, PfPK6, a kinase with sequence features characteristic of both a CDK and MAP kinase, is unaffected by this CDI. Furthermore, the CDK4/6 specific CDI, p16(INK4), fails to inhibit these plasmodial CDKs. Taken together, these results suggest that plasmodial CDKs may be regulated by the binding of inhibitory proteins in vivo.

New class of small nonpeptidyl compounds blocks Plasmodium falciparum development in vitro by inhibiting plasmepsins.

Malarial parasites rely on aspartic proteases called plasmepsins to digest hemoglobin during the intraerythrocytic stage. Plasmepsins from Plasmodium falciparum and Plasmodium vivax have been cloned and expressed for a variety of structural and enzymatic studies. Recombinant plasmepsins possess kinetic similarity to the native enzymes, indicating their suitability for target-based antimalarial drug development. We developed an automated assay of P. falciparum plasmepsin II and P. vivax plasmepsin to quickly screen compounds in the Walter Reed chemical database. A low-molecular-mass (346 Da) diphenylurea derivative (WR268961) was found to inhibit plasmepsins with a K(i) of 1 to 6 microM. This compound appears to be selective for plasmepsin, since it is a poor inhibitor of the human aspartic protease cathepsin D (K(i) greater than 280 microM). WR268961 inhibited the growth of P. falciparum strains W2 and D6, with 50% inhibitory concentrations ranging from 0.03 to 0.16 microg/ml, but was much less toxic to mammalian cells. The Walter Reed chemical database contains over 1,500 compounds with a diphenylurea core structure, 9 of which inhibit the plasmepsins, with K(i) values ranging from 0.05 to 0.68 microM. These nine compounds show specificity for the plasmepsins over human cathepsin D, but they are poor inhibitors of P. falciparum growth in vitro. Computational docking experiments indicate how diphenylurea compounds bind to the plasmepsin active site and inhibit the enzyme.

New insights into copper monooxygenases and peptide amidation: structure, mechanism and function.

Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-C alpha bond. In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all the peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5), generates alpha-amidated peptide product and glyoxylate. PHM contains two redox-active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. The structure of the catalytic core of rat PHM at atomic resolution provides a framework for understanding the broad substrate specificity of PHM, identifying residues critical for PHM activity, and proposing mechanisms for the chemical and electron-transfer steps in catalysis. Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase (DBM; EC 1.14.17.1), the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM.

Cyclin H activation and drug susceptibility of the Pfmrk cyclin dependent protein kinase from Plasmodium falciparum.

The eukaryotic cell cycle is regulated by a group of highly conserved cyclin dependent protein kinases (CDKs). Several CDKs have been identified in Plasmodium falciparum, however, their regulatory mechanisms as well as their role in parasite growth and differentiation are not understood fully. To further our understanding of Plasmodium CDK regulation, we have characterized Pfmrk kinase activity. Pfmrk was expressed and purified as a 6xHis tagged recombinant protein from Escherichia coli and assayed for histone H1 kinase activity. Pfmrk has significant histone H1 kinase activity and is autophosphorylated in vitro. Human cyclin H forms a stable complex with Pfmrk and stimulates kinase activity. This is the first indication that Plasmodial CDKs are partially regulated by cyclin subunits, as are human CDKs. CDKs are attractive drug targets due to their role in cellular proliferation. Specific CDK inhibitors were selected to evaluate Pfmrk as a potential drug target. Olomoucine and roscovitine failed to inhibit Pfmrk kinase activity which places Pfmrk with a class of CDKs that are insensitive to these compounds. A molecular model of Pfmrk provides a structural explanation for the failure of these compounds to inhibit Pfmrk.

Molecular characterization of Plasmodium falciparum S-adenosylmethionine synthetase.

S-Adenosylmethionine (AdoMet) synthetase (SAMS: EC 2.5.1.6) catalyses the formation of AdoMet from methionine and ATP. We have cloned a gene for Plasmodium falciparum AdoMet synthetase (PfSAMS) (GenBank accession no. AF097923), consisting of 1209 base pairs with no introns. The gene encodes a polypeptide (PfSAMS) of 402 amino acids with a molecular mass of 44844 Da, and has an overall base composition of 67% A+T. PfSAMS is probably a single copy gene, and was mapped to chromosome 9. The PfSAMS protein is highly homologous to all other SAMS, including a conserved motif for the phosphate-binding P-loop, HGGGAFSGKD, and the signature hexapeptide, GAGDQG. All the active-site amino acids for the binding of ADP, P(i) and metal ions are similarly preserved, matching entirely those of human hepatic SAMS and Escherichia coli SAMS. Molecular modelling of PfSAMS guided by the X-ray crystal structure of E. coli SAMS indicates that PfSAMS binds ATP/Mg(2+) in a manner similar to that seen in the E. coli SAMS structure. However, the PfSAMS model shows that it can not form tetramers as does E. coli SAMS, and is probably a dimer instead. There was a differential sensitivity towards the inhibition by cycloleucine between the expressed PfSAMS and the human hepatic SAMS with K(i) values of 17 and 10 mM, respectively. Based on phylogenetic analysis using protein parsimony and neighbour-joining algorithms, the malarial PfSAMS is closely related to SAMS of other protozoans and plants.

Substrate-mediated electron transfer in peptidylglycine alpha-hydroxylating monooxygenase.

Peptide amidation is a ubiquitous posttranslational modification of bioactive peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first step of this reaction, is composed of two domains, each of which binds one copper atom. The coppers are held 11 A apart on either side of a solvent-filled interdomain cleft, and the PHM reaction requires electron transfer between these sites. A plausible mechanism for electron transfer might involve interdomain motion to decrease the distance between the copper atoms. Our experiments show that PHM catalytic core (PHMcc) is enzymatically active in the crystal phase, where interdomain motion is not possible. Instead, structures of two states relevant to catalysis indicate that water, substrate and active site residues may provide an electron transfer pathway that exists only during the PHM catalytic cycle.

Amidation of bioactive peptides: the structure of peptidylglycine alpha-hydroxylating monooxygenase.

Many neuropeptides and peptide hormones require amidation at the carboxyl terminus for activity. Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of these diverse physiological regulators. The amino-terminal domain of the bifunctional PAM protein is a peptidylglycine alpha-hydroxylating monooxygenase (PHM) with two coppers that cycle through cupric and cuprous oxidation states. The anomalous signal of the endogenous coppers was used to determine the structure of the catalytic core of oxidized rat PHM with and without bound peptide substrate. These structures strongly suggest that the PHM reaction proceeds via activation of substrate by a copper-bound oxygen species. The mechanistic and structural insight gained from the PHM structures can be directly extended to dopamine beta-monooxygenase.

Structure and mechanism of lipoxygenases.

In mammals, lipoxygenases catalyze the formation of hydroperoxides as the first step in the biosynthesis of several inflammatory mediators. The substrate of this reaction, arachidonic acid, is the key precursor of two families of potent physiological effectors. It is the branch point between two central pathways: one, involving the enzyme cyclooxygenase, leads to the synthesis of prostaglandins and thromboxanes; the other, involving lipoxygenases, leads to the synthesis of leukotrienes and lipoxins, compounds that regulate important cellular responses in inflammation and immunity. While aspirin and other non-steroidal anti-inflammatory compounds are potent inhibitors of cyclooxygenase, no effective pharmacological inhibitor of lipoxygenase is presently available. Lipoxygenases are large non-heme, iron-containing enzymes that use molecular oxygen for the diooxygenation of arachidonic acid to form hydroperoxides, the first step in the biosynthetic pathways leading to leukotrienes and lipoxins. Because of the importance of these compounds, lipoxygenases have been the subject of extensive study: from detailed kinetic measurements to cloning, expression, and site-directed mutagenesis. The sequences of over 50 lipoxygenases have been reported. In addition, the structure of soybean lipoxygenase-1, determined by X-ray diffraction methods, has recently been reported. The structure revealed that the 839 amino acids in the protein are organized in two domains: a beta-sheet N-terminal domain and a large, mostly helical C-terminal domain. The iron is present in the C-terminal domain facing two internal cavities that are probably the conduits through which the fatty acid and molecular oxygen gain access to the metal. Models of the mammalian lipoxygenases based on the soybean structure provide clues about the structural determinants of the positional specificity of the enzyme, and can be used as targets for the design of more effective inhibitors.

Structure conservation in lipoxygenases: structural analysis of soybean lipoxygenase-1 and modeling of human lipoxygenases.

Lipoxygenases are a class of non-heme iron dioxygenases which catalyze the hydroperoxidation of fatty acids for the biosynthesis of leukotrienes and lipoxins. The structure of the 839-residue soybean lipoxygenase-1 was used as a template to model human 5-, 12-, and 15-lipoxygenases. A distance-based algorithm for placing side chains in a low homology environment (only the four iron ligands were fixed during side chain placement) was devised. Twenty-six of the 56 conserved lipoxygenase residues were grouped in four distinct regions of the enzyme. These regions were analyzed to discern whether the side chain interactions could be duplicated in the models or whether alternate conformers should be considered. The effects of site directed mutagenesis variants were rationalized using the models of the human lipoxygenases. In particular, variants which shifted positional specificity between 12- and 15-lipoxygenase activity were analyzed. Analysis of active site residues produced a model which accounts for observed lipoxygenase positional specificity and stereospecificity.