RESUMO
Trypanosoma brucei prostaglandin F2alpha synthase is an aldo-ketoreductase that catalyzes the reduction of prostaglandin H2 to PGF2alpha in addition to that of 9,10-phenanthrenequinone. We report the crystal structure of TbPGFS.NADP+.citrate at 2.1 angstroms resolution. TbPGFS adopts a parallel (alpha/beta)8-barrel fold lacking the protrudent loops and possesses a hydrophobic core active site that contains a catalytic tetrad of tyrosine, lysine, histidine, and aspartate, which is highly conserved among AKRs. Site-directed mutagenesis of the catalytic tetrad residues revealed that a dyad of Lys77 and His110, and a triad of Tyr52, Lys77, and His110 are essential for the reduction of PGH2 and 9,10-PQ, respectively. Structural and kinetic analysis revealed that His110, acts as the general acid catalyst for PGH2 reduction and that Lys77 facilitates His110 protonation through a water molecule, while exerting an electrostatic repulsion against His110 that maintains the spatial arrangement which allows the formation of a hydrogen bond between His110 and C11 that carbonyl of PGH2. We also show Tyr52 acts as the general acid catalyst for 9,10-PQ reduction, and thus we not only elucidate the catalytic mechanism of a PGH2 reductase but also provide an insight into the catalytic specificity of AKRs.
Assuntos
Hidroxiprostaglandina Desidrogenases/química , Hidroxiprostaglandina Desidrogenases/genética , Oxirredutases/metabolismo , Prostaglandina H2/química , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Domínio Catalítico , Dicroísmo Circular , Citratos/química , Cristalografia por Raios X , Análise Mutacional de DNA , DNA Complementar/metabolismo , Evolução Molecular , Humanos , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Prótons , Ratos , Homologia de Sequência de Aminoácidos , Suínos , Tirosina/química , Raios UltravioletaRESUMO
Human African trypanosomiasis is undergoing an alarming rate of recrudescence in many parts of sub-Saharan Africa. Yet, there is no successful chemotherapy for the disease due to a limited number of useful drugs, side effects and drawbacks of the existing medication, as well as the development of drug resistance by the parasite. Here we describe a new lead anti-trypanosomal compound isolated from Kola acuminata (Makasu). We purified a proanthocyanidin by chromatographic procedures and confirmed its homogeneity and structure by Nuclear Magnetic Resonance and Matrix-Assisted Laser Desorption Ionisation Time-of-Flight mass spectrometry, respectively. In vitro, this compound potently induced growth arrest and lysis of bloodstream form trypanosomes in a dose- and time-dependent manner. In a mouse model, it exhibited a trypanostatic effect that extended the life of infected, treated animals up to 8 days post-infection against the 4 days for infected, untreated animals. The proanthocyanidin showed a low cytotoxicity against mammalian cells, whereas treated-BF showed massive enlargement of their flagellar pocket and lysosome-like structures caused by an intense formation of multivesicular bodies and vesicles within these organelles. The observed ultrastructural alterations caused rupture of plasma membranes and the release of cell contents, indicative of a necrotic process rather than a programmed cell death. Interestingly, the proanthocyanidin acted against BF but not procyclic form trypanosomes. This new anti-trypanosomal compound should be further studied to determine its efficacy and suitability as an anti-trypanosomal drug and may be used as a tool to define novel specific drug targets in BF trypanosomes.
Assuntos
Cola , Fitoterapia/métodos , Proantocianidinas/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Animais , Cromatografia em Camada Fina/métodos , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Proantocianidinas/química , Proantocianidinas/isolamento & purificação , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/ultraestrutura , Células Tumorais CultivadasRESUMO
Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.