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Takashi Aoki - One of the best experts on this subject based on the ideXlab platform.
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structures of trypanosoma cruzi Dihydroorotate dehydrogenase complexed with substrates and products atomic resolution insights into mechanisms of Dihydroorotate oxidation and fumarate reduction
Biochemistry, 2008Co-Authors: Daniel Ken Inaoka, Takeshi Nara, Takashi Aoki, Kimitoshi Sakamoto, Hironari Shimizu, Tomoo Shiba, Genji Kurisu, Kiyoshi Kita, Shigeharu HaradaAbstract:Dihydroorotate dehydrogenase (DHOD) from Trypanosoma cruzi (TcDHOD) is a member of family 1A DHOD that catalyzes the oxidation of Dihydroorotate to orotate (first half-reaction) and then the reduction of fumarate to succinate (second half-reaction) in the de novo pyrimidine biosynthesis pathway. The oxidation of Dihydroorotate is coupled with the reduction of FMN, and the reduced FMN converts fumarate to succinate in the second half-reaction. TcDHOD are known to be essential for survival and growth of T. cruzi and a validated drug target. The first-half reaction mechanism of the family 1A DHOD from Lactococcus lactis has been extensively investigated on the basis of kinetic isotope effects, mutagenesis and X-ray structures determined for ligand-free form and in complex with orotate, the product of the first half-reaction. In this report, we present crystal structures of TcDHOD in the ligand-free form and in complexes with an inhibitor, physiological substrates and products of the first and second half-rea...
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expression purification and crystallization of trypanosoma cruzi Dihydroorotate dehydrogenase complexed with orotate
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005Co-Authors: Daniel Ken Inaoka, Takashi Aoki, Hironari Shimizu, Shigeharu Harada, Eizo Takashima, Arihiro Osanai, T Nara, Kiyoshi KitaAbstract:Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of Dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 A resolution using synchrotron radiation (λ = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall R merge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V M) of 2.2 A3 Da−1 and a solvent content of 44%.
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inhibitory action of marine algae extracts on the trypanosoma cruzi Dihydroorotate dehydrogenase activity and on the protozoan growth in mammalian cells
Parasitology International, 2005Co-Authors: Takeshi Nara, Yuto Kamei, Akiko Tsubouchi, Takeshi Annoura, Kenichiro Hirota, Kyoichi Iizumi, Yuki Dohmoto, Takeaki Ono, Takashi AokiAbstract:Abstract Trypanosoma cruzi, the causative agent of Chagas' disease, replicates in mammalian cells and relies on the de novo pyrimidine biosynthetic pathway that supplies essential precursors for nucleic acid synthesis. The protozoan Dihydroorotate dehydrogenase (DHOD), the fourth enzyme of the pathway catalyzing production of orotate from Dihydroorotate, markedly differs from the human enzyme. This study was thus aimed to search for potent inhibitors against T. cruzi DHOD activity, and a number of methanol extracts prepared from green, brown, and red algae were assayed. The extracts from two brown algae, Fucus evanescens and Pelvetia babingtonii, yielded 59 and 58% decrease in the recombinant DHOD activity, respectively, at the concentration of 50 μg/ml. Inhibition by these extracts was noncompetitive with respect to Dihydroorotate, with apparent Ki values of 35.3±5.9 and 10.3±4.4 μg/ml, respectively. Further, in an in vitro T. cruzi–HeLa cell infection system, ethanol-reconstituted F. evanescens and P. babingtonii extracts at the concentration of 1 μg/ml, respectively, decreased significantly the infection rate of host cells and the average parasite number per infected cell. These results imply that F. evanescens and P. babingtonii contain inhibitor(s) against the T. cruzi DHOD activity and against the protozoan infection and proliferation in mammalian cells. Identification of inhibitor(s) in these two brown algae and further screening of other marine algae may facilitate the discovery of new, anti-trypanosomal lead compounds.
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novel organization and sequences of five genes encoding all six enzymes for de novo pyrimidine biosynthesis in trypanosoma cruzi
Journal of Molecular Biology, 1999Co-Authors: Takeshi Nara, Junko Nakajimashimada, Takashi AokiAbstract:A 25 kb segment of genomic DNA from Trypanosoma cruzi, the causative agent of Chagas’ disease, was sequenced. It contains five genes, pyr1, pyr2, pyr3, pyr4, and pyr6–5, encoding all six enzymes involved in de novo pyrimidine biosynthesis, glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydroorotase, Dihydroorotate dehydrogenase, and orotidine-5′-phosphate decarboxylase linked with orotate phosphoribosyltransferase, respectively. The pyr genes constitute a polycistronic transcription unit on an 800 kb chromosomal DNA in the order of pyr1, pyr3, pyr6–5, pyr2, and pyr4 from the 5′ terminus, with intervening sequences of 2.2, 0.4, 8.1, and 0.8 kb. The amino acid sequences deduced from the trypanosomatid pyr genes, except for pyr6, showed closer similarities to mammalian and yeast sequences, and less similarity to archaeal and bacterial sequences. The last two enzymes encoded by a single gene, pyr6–5, are covalently linked in the order opposite to mammalian pyr5–6, and possess a putative glycosomal targeting signal tripeptide, serine-lysine-leucine, at the C terminus. The calculated isoelectric points of 9.3 and 9.9 are also diagnostic of the glycosomal localization of these enzymes. We conclude that the T. cruzi pyr gene organization represents an early progenitor in de novo pyrimidine biosynthesis in eukaryotic lineage, and that the independent pyr genes may have evolved before the gene fusion events that resulted in the three mammalian-type genes, pyr1–3–2, pyr4, and pyr5–6, for UMP synthesis. Peculiarities in thetrypanosomatid pyr6–5 gene product are discussed.
Takeshi Nara - One of the best experts on this subject based on the ideXlab platform.
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structures of trypanosoma cruzi Dihydroorotate dehydrogenase complexed with substrates and products atomic resolution insights into mechanisms of Dihydroorotate oxidation and fumarate reduction
Biochemistry, 2008Co-Authors: Daniel Ken Inaoka, Takeshi Nara, Takashi Aoki, Kimitoshi Sakamoto, Hironari Shimizu, Tomoo Shiba, Genji Kurisu, Kiyoshi Kita, Shigeharu HaradaAbstract:Dihydroorotate dehydrogenase (DHOD) from Trypanosoma cruzi (TcDHOD) is a member of family 1A DHOD that catalyzes the oxidation of Dihydroorotate to orotate (first half-reaction) and then the reduction of fumarate to succinate (second half-reaction) in the de novo pyrimidine biosynthesis pathway. The oxidation of Dihydroorotate is coupled with the reduction of FMN, and the reduced FMN converts fumarate to succinate in the second half-reaction. TcDHOD are known to be essential for survival and growth of T. cruzi and a validated drug target. The first-half reaction mechanism of the family 1A DHOD from Lactococcus lactis has been extensively investigated on the basis of kinetic isotope effects, mutagenesis and X-ray structures determined for ligand-free form and in complex with orotate, the product of the first half-reaction. In this report, we present crystal structures of TcDHOD in the ligand-free form and in complexes with an inhibitor, physiological substrates and products of the first and second half-rea...
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inhibitory action of marine algae extracts on the trypanosoma cruzi Dihydroorotate dehydrogenase activity and on the protozoan growth in mammalian cells
Parasitology International, 2005Co-Authors: Takeshi Nara, Yuto Kamei, Akiko Tsubouchi, Takeshi Annoura, Kenichiro Hirota, Kyoichi Iizumi, Yuki Dohmoto, Takeaki Ono, Takashi AokiAbstract:Abstract Trypanosoma cruzi, the causative agent of Chagas' disease, replicates in mammalian cells and relies on the de novo pyrimidine biosynthetic pathway that supplies essential precursors for nucleic acid synthesis. The protozoan Dihydroorotate dehydrogenase (DHOD), the fourth enzyme of the pathway catalyzing production of orotate from Dihydroorotate, markedly differs from the human enzyme. This study was thus aimed to search for potent inhibitors against T. cruzi DHOD activity, and a number of methanol extracts prepared from green, brown, and red algae were assayed. The extracts from two brown algae, Fucus evanescens and Pelvetia babingtonii, yielded 59 and 58% decrease in the recombinant DHOD activity, respectively, at the concentration of 50 μg/ml. Inhibition by these extracts was noncompetitive with respect to Dihydroorotate, with apparent Ki values of 35.3±5.9 and 10.3±4.4 μg/ml, respectively. Further, in an in vitro T. cruzi–HeLa cell infection system, ethanol-reconstituted F. evanescens and P. babingtonii extracts at the concentration of 1 μg/ml, respectively, decreased significantly the infection rate of host cells and the average parasite number per infected cell. These results imply that F. evanescens and P. babingtonii contain inhibitor(s) against the T. cruzi DHOD activity and against the protozoan infection and proliferation in mammalian cells. Identification of inhibitor(s) in these two brown algae and further screening of other marine algae may facilitate the discovery of new, anti-trypanosomal lead compounds.
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novel organization and sequences of five genes encoding all six enzymes for de novo pyrimidine biosynthesis in trypanosoma cruzi
Journal of Molecular Biology, 1999Co-Authors: Takeshi Nara, Junko Nakajimashimada, Takashi AokiAbstract:A 25 kb segment of genomic DNA from Trypanosoma cruzi, the causative agent of Chagas’ disease, was sequenced. It contains five genes, pyr1, pyr2, pyr3, pyr4, and pyr6–5, encoding all six enzymes involved in de novo pyrimidine biosynthesis, glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydroorotase, Dihydroorotate dehydrogenase, and orotidine-5′-phosphate decarboxylase linked with orotate phosphoribosyltransferase, respectively. The pyr genes constitute a polycistronic transcription unit on an 800 kb chromosomal DNA in the order of pyr1, pyr3, pyr6–5, pyr2, and pyr4 from the 5′ terminus, with intervening sequences of 2.2, 0.4, 8.1, and 0.8 kb. The amino acid sequences deduced from the trypanosomatid pyr genes, except for pyr6, showed closer similarities to mammalian and yeast sequences, and less similarity to archaeal and bacterial sequences. The last two enzymes encoded by a single gene, pyr6–5, are covalently linked in the order opposite to mammalian pyr5–6, and possess a putative glycosomal targeting signal tripeptide, serine-lysine-leucine, at the C terminus. The calculated isoelectric points of 9.3 and 9.9 are also diagnostic of the glycosomal localization of these enzymes. We conclude that the T. cruzi pyr gene organization represents an early progenitor in de novo pyrimidine biosynthesis in eukaryotic lineage, and that the independent pyr genes may have evolved before the gene fusion events that resulted in the three mammalian-type genes, pyr1–3–2, pyr4, and pyr5–6, for UMP synthesis. Peculiarities in thetrypanosomatid pyr6–5 gene product are discussed.
Shigeharu Harada - One of the best experts on this subject based on the ideXlab platform.
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pharmacophore modeling for anti chagas drug design using the fragment molecular orbital method
PLOS ONE, 2015Co-Authors: Ryunosuke Yoshino, Daniel Ken Inaoka, Tomoo Shiba, Shigeharu Harada, Nobuaki Yasuo, Yohsuke Hagiwara, Kazuki Ohno, Masaya Orita, Masayuki Inoue, Teruki HonmaAbstract:Background Chagas disease, caused by the parasite Trypanosoma cruzi, is a neglected tropical disease that causes severe human health problems. To develop a new chemotherapeutic agent for the treatment of Chagas disease, we predicted a pharmacophore model for T. cruzi Dihydroorotate dehydrogenase (TcDHODH) by fragment molecular orbital (FMO) calculation for orotate, oxonate, and 43 orotate derivatives.
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structures of trypanosoma cruzi Dihydroorotate dehydrogenase complexed with substrates and products atomic resolution insights into mechanisms of Dihydroorotate oxidation and fumarate reduction
Biochemistry, 2008Co-Authors: Daniel Ken Inaoka, Takeshi Nara, Takashi Aoki, Kimitoshi Sakamoto, Hironari Shimizu, Tomoo Shiba, Genji Kurisu, Kiyoshi Kita, Shigeharu HaradaAbstract:Dihydroorotate dehydrogenase (DHOD) from Trypanosoma cruzi (TcDHOD) is a member of family 1A DHOD that catalyzes the oxidation of Dihydroorotate to orotate (first half-reaction) and then the reduction of fumarate to succinate (second half-reaction) in the de novo pyrimidine biosynthesis pathway. The oxidation of Dihydroorotate is coupled with the reduction of FMN, and the reduced FMN converts fumarate to succinate in the second half-reaction. TcDHOD are known to be essential for survival and growth of T. cruzi and a validated drug target. The first-half reaction mechanism of the family 1A DHOD from Lactococcus lactis has been extensively investigated on the basis of kinetic isotope effects, mutagenesis and X-ray structures determined for ligand-free form and in complex with orotate, the product of the first half-reaction. In this report, we present crystal structures of TcDHOD in the ligand-free form and in complexes with an inhibitor, physiological substrates and products of the first and second half-rea...
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expression purification and crystallization of trypanosoma cruzi Dihydroorotate dehydrogenase complexed with orotate
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005Co-Authors: Daniel Ken Inaoka, Takashi Aoki, Hironari Shimizu, Shigeharu Harada, Eizo Takashima, Arihiro Osanai, T Nara, Kiyoshi KitaAbstract:Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of Dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 A resolution using synchrotron radiation (λ = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall R merge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V M) of 2.2 A3 Da−1 and a solvent content of 44%.
Kaj Frank Jensen - One of the best experts on this subject based on the ideXlab platform.
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multiple states of the tyr318leu mutant of Dihydroorotate dehydrogenase revealed by single molecule kinetics
Journal of the American Chemical Society, 2004Co-Authors: Jue Shi, Kaj Frank Jensen, Bruce A Palfey, Joe Dertouzos, Ari Gafni, D G SteelAbstract:Dihydroorotate dehydrogenase (DHOD) from Escherichia coli is a monomeric membrane-associated flavoprotein that catalyzes the oxidation of Dihydroorotate to orotate. By using confocal fluorescence spectroscopy on the highly fluorescent Tyr318Leu DHOD mutant, we studied the catalytic turnover of single enzyme molecules through the characteristic on-off fluorescence signal, which corresponds to flavin mononucleotide (FMN) interconverting between the oxidized and reduced states during turnover. Our single-molecule data provide evidence of a distinct static heterogeneity in the enzymatic activity, with some molecules going through the on-off cycles 5-fold faster than others, however, there is no detectable dynamic disorder in DHOD turnover. When 0.1% reduced Triton X-100, a detergent that more closely simulates the natural membrane environment, is added, our data suggest the degree of static molecular heterogeneity is reduced. The observation of static heterogeneity suggests that the enzyme, which associates with the membrane in vivo, is present in distinct conformations that result in different catalytic efficiencies. The alternate conformations are most likely the result of the loss of van der Waals or other interactions between tyrosine 318 and FMN in the catalytic site with the mutation of Tyr318Leu, which disrupts the native structure of wild-type DHOD.
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E. coli Dihydroorotate Dehydrogenase Reveals Structural and Functional Distinctions between Different Classes of Dihydroorotate Dehydrogenases
Structure (London England : 1993), 2002Co-Authors: Sofie Nørager, Olof Bjornberg, Kaj Frank Jensen, Sine LarsenAbstract:The flavoenzymes Dihydroorotate dehydrogenases (DHODs) catalyze the fourth and only redox step in the de novo biosynthesis of UMP. Enzymes belonging to class 2, according to their amino acid sequence, are characterized by having a serine residue as the catalytic base and a longer N terminus. The structure of class 2 E. coli DHOD, determined by MAD phasing, showed that the N-terminal extension forms a separate domain. The catalytic serine residue has an environment differing from the equivalent cysteine in class 1 DHODs. Significant differences between the two classes of DHODs were identified by comparison of the E. coli DHOD with the other known DHOD structures, and differences with the class 2 human DHOD explain the variation in their inhibitors.
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insight into the chemistry of flavin reduction and oxidation in escherichia coli Dihydroorotate dehydrogenase obtained by rapid reaction studies
Biochemistry, 2001Co-Authors: Bruce A Palfey, Olof Bjornberg, Kaj Frank JensenAbstract:Dihydroorotate dehydrogenase (DHOD) oxidizes Dihydroorotate (DHO) to orotate in the only redox reaction of pyrimidine biosynthesis. The enzyme from Escherichia coli is a membrane-bound FMN-containing enzyme that is thought to use ubiquinone as the oxidizing substrate. The chemistry of the reduction of the flavin in DHOD from E. coli by the substrate Dihydroorotate (DHO) was studied at 4 degrees C in anaerobic stopped-flow experiments conducted over a broad range of pH values. A Michaelis complex that was characterized by a approximately 20 nm red-shift of the oxidized flavin absorbance formed within the dead-time of the stopped-flow instrument ( approximately 1 ms) upon mixing with DHO. The flavin of the intermediate was reduced by DHO, forming a reduced flavin-orotate charge-transfer complex. The rate constant for the flavin reduction reaction increased with pH, from a value of 1 s(-1) at pH 6.5 to approximately 360 s(-1) at pH values greater than an observed pK(a) of 9.5 which was ascribed to Ser175, the active-site base. At all pH values, the reduced flavin-orotate charge-transfer complex dissociated too slowly to be catalytically relevant. Therefore, the oxidizing quinone substrate must bind to the reduced enzyme-orotate complex at a site distinct from the substrate binding site, in agreement with steady-state kinetic studies [Bjornberg, O., Gruner, A.-C., Roepstorff, P., and Jensen, K. F. (1999) Biochemistry 38, 2899-2908]. Menadione was used as a model quinone substrate to oxidize dithionite-reduced DHOD. The reduced enzyme-orotate complex reacted rapidly with menadione (180 s(-1)), demonstrating that the reduced enzyme-orotate complex is a catalytically competent intermediate.
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the activity of escherichia coli Dihydroorotate dehydrogenase is dependent on a conserved loop identified by sequence homology mutagenesis and limited proteolysis
Biochemistry, 1999Co-Authors: Olof Bjornberg, Annecharlotte Gruner, Peter Roepstorff, Kaj Frank JensenAbstract:Dihydroorotate dehydrogenase catalyzes the oxidation of Dihydroorotate to orotate. The enzyme from Escherichia coli was overproduced and characterized in comparison with the dimeric Lactococcus lactis A enzyme, whose structure is known. The two enzymes represent two distinct evolutionary families of Dihydroorotate dehydrogenases, but sedimentation in sucrose gradients suggests a dimeric structure also of the E. coli enzyme. Product inhibition showed that the E. coli enzyme, in contrast to the L. lactis enzyme, has separate binding sites for Dihydroorotate and the electron acceptor. Trypsin readily cleaved the E. coli enzyme into two fragments of 182 and 154 residues, respectively. Cleavage reduced the activity more than 100-fold but left other molecular properties, including the heat stability, intact. The trypsin cleavage site, at R182, is positioned in a conserved region that, in the L. lactis enzyme, forms a loop where a cysteine residue is very critical for activity. In the corresponding position, the...
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the crystal structure of lactococcus lactis Dihydroorotate dehydrogenase a complexed with the enzyme reaction product throws light on its enzymatic function
Protein Science, 1998Co-Authors: Paul Rowland, Olof Bjornberg, Kaj Frank Jensen, Finn S Nielsen, Sine LarsenAbstract:Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-Dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lactococcus lactis Dihydroorotate dehydrogenase A (DHODA) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 A resolution has been compared with the structure of the native enzyme. The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA flavin isoalloxazine ring, causing only small movements of the surrounding protein residues. The orotate is completely buried beneath the protein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop. The orotate is bound by four conserved asparagine side chains (Asn 67, Asn 127, Asn 132, and Asn 193), the side chains of Lys 43 and Ser 194, and the main chain NH groups of Met 69, Gly 70, and Leu 71. Of these the Lys 43 side chain makes hydrogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate. Potential interactions with bound Dihydroorotate are considered using the orotate complex as a basis for molecular modeling. The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the different families of DHODs is reviewed, along with implications for differences in substrate binding and in the catalytic mechanisms between these families.
Daniel Ken Inaoka - One of the best experts on this subject based on the ideXlab platform.
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pharmacophore modeling for anti chagas drug design using the fragment molecular orbital method
PLOS ONE, 2015Co-Authors: Ryunosuke Yoshino, Daniel Ken Inaoka, Tomoo Shiba, Shigeharu Harada, Nobuaki Yasuo, Yohsuke Hagiwara, Kazuki Ohno, Masaya Orita, Masayuki Inoue, Teruki HonmaAbstract:Background Chagas disease, caused by the parasite Trypanosoma cruzi, is a neglected tropical disease that causes severe human health problems. To develop a new chemotherapeutic agent for the treatment of Chagas disease, we predicted a pharmacophore model for T. cruzi Dihydroorotate dehydrogenase (TcDHODH) by fragment molecular orbital (FMO) calculation for orotate, oxonate, and 43 orotate derivatives.
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structures of trypanosoma cruzi Dihydroorotate dehydrogenase complexed with substrates and products atomic resolution insights into mechanisms of Dihydroorotate oxidation and fumarate reduction
Biochemistry, 2008Co-Authors: Daniel Ken Inaoka, Takeshi Nara, Takashi Aoki, Kimitoshi Sakamoto, Hironari Shimizu, Tomoo Shiba, Genji Kurisu, Kiyoshi Kita, Shigeharu HaradaAbstract:Dihydroorotate dehydrogenase (DHOD) from Trypanosoma cruzi (TcDHOD) is a member of family 1A DHOD that catalyzes the oxidation of Dihydroorotate to orotate (first half-reaction) and then the reduction of fumarate to succinate (second half-reaction) in the de novo pyrimidine biosynthesis pathway. The oxidation of Dihydroorotate is coupled with the reduction of FMN, and the reduced FMN converts fumarate to succinate in the second half-reaction. TcDHOD are known to be essential for survival and growth of T. cruzi and a validated drug target. The first-half reaction mechanism of the family 1A DHOD from Lactococcus lactis has been extensively investigated on the basis of kinetic isotope effects, mutagenesis and X-ray structures determined for ligand-free form and in complex with orotate, the product of the first half-reaction. In this report, we present crystal structures of TcDHOD in the ligand-free form and in complexes with an inhibitor, physiological substrates and products of the first and second half-rea...
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expression purification and crystallization of trypanosoma cruzi Dihydroorotate dehydrogenase complexed with orotate
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005Co-Authors: Daniel Ken Inaoka, Takashi Aoki, Hironari Shimizu, Shigeharu Harada, Eizo Takashima, Arihiro Osanai, T Nara, Kiyoshi KitaAbstract:Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of Dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 A resolution using synchrotron radiation (λ = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall R merge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V M) of 2.2 A3 Da−1 and a solvent content of 44%.