Old yellow enzyme (OYE) is an NADPH oxidoreductase that contains a flavin mononucleotide as a prosthetic group. The OYE from Trypanosoma cruzi, which produces prostaglandin F(2alpha), a potent mediator of various physiological and pathological processes, from prostaglandin H2. The protein was recombinantly expressed and purified from Escherichia coli and was crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 56.3, b = 78.8, c = 78.8 A, beta = 93.4 degrees and two molecules per asymmetric unit. The crystals were suitable for X-ray crystallographic studies and diffracted to 1.70 A resolution. A Patterson search method is in progress using the structure of OYE from Pseudomonas putida as a starting model.

Trypanosoma brucei prostaglandin (PG) F 2 synthase (TbPGFS), an aldo-keto reductase, catalyzes the NADPH-dependent reduction of the endoperoxide moiety of PGH 2 to PGF 2 .The overproduction of PGF 2 during trypanosomasis causes miscarriage in infected subjects.Here we report the crystal structures of TbPGFS-NADP + bounds citrate or sulfate at 2.1 Å and 2.6 Å resolution respectively.TbPGFS adopts a parallel ( / ) 8 -barrel folds lacking the protrudent loops.The core active site structure is hydrophobic to bind hydrophobic substrates and contains tyrosine, lysine, histidine and aspartate known as a catalytic tetrad which is preserved in most of other aldo-keto reductases.These four residues are said to be indispensable for the reduction of PGH 2 , but mutagenesis shows that Tyr52 and Asp47 are not involved in the enzyme reaction and identifies His110 and Lys77 work as catalytic dyad.His 110 acts as a general acid catalyst, while Lys 77 facilitates proton donation by His 110 through a water molecule and forms a salt-bridge to stabilize the Asp 47 that binds NADPH.By comparing the citrate and sulfate complex structure, we detected that Trp187 holds the nicotinamide ring of NADPH from tilting on the access of PGH 2 .These findings reveal a novel catalytic mechanism for the biological reduction of the endoperoxide PGH 2 by an aldo-keto reductase.The structure should allow for rational design of specific inhibitors useful to investigate the physiological roles of TbPGFS in trypanosomes.

Trypanosoma brucei prostaglandin F2α synthase is an aldo-ketoreductase that catalyzes the reduction of prostaglandin H2 to PGF2α in addition to that of 9,10-phenanthrenequinone. We report the crystal structure of TbPGFS·NADP+·citrate at 2.1 Å resolution. TbPGFS adopts a parallel (α/β)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.

In mast and Th2 cells, hematopoietic prostaglandin (PG) D synthase (H-PGDS) catalyses the isomerization of PGH(2) in the presence of glutathione (GSH) to produce the allergic and inflammatory mediator PGD(2). We determined the X-ray structures of human H-PGDS inhibitor complexes with 1-amino-4-{4-[4-chloro-6-(2-sulpho-phenylamino)-[1,3,5]triazin-2-ylmethyl]-3-sulpho-phenylamino}-9,10-dioxo-9,10-dihydro-anthracene-2-sulphonic acid (Cibacron Blue) and 1-amino-4-(4-aminosulphonyl) phenyl-anthraquinone-2-sulphonic acid (APAS) at 2.0 Å resolution. When complexed with H-PGDS, Cibacron Blue had an IC(50) value of 40 nM and APAS 2.1 μM. The Cibacron Blue molecule was stabilized by four hydrogen bonds and π-π stacking between the anthraquinone ring and Trp104, the ceiling of the active site H-PGDS pocket. Among the four hydrogen bonds, the Cibacron Blue terminal sulphonic group directly interacted with conserved residues Lys112 and Lys198, which recognize the PGH(2) substrate α-chain. In contrast, the APAS anthraquinone ring was inverted to interact with Trp104, while its benzenesulphonic group penetrated the GSH-bound region at the bottom of the active site. Due to the lack of extended aromatic rings, APAS could not directly hydrogen bond with the two conserved lysine residues, thus decreasing the total number of hydrogen bond from four to one. These factors may contribute to the 50-fold difference in the IC(50) values obtained for the two inhibitors.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyzes the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation cycles by combining CO2 and O2, respectively, with ribulose-1,5-bisphosphate. Many photosynthetic organisms have form I rubiscos comprised of eight large (L) and eight small (S) subunits. The crystal structure of the complex of activated rubisco from the green alga Chlamydomonas reinhardtii and the reaction intermediate analogue 2-carboxyarabinitol-1,5-bisphosphate (2-CABP) has been solved at 1.84 Å resolution (Rcryst of 15.2 % and Rfree of 18.1 %). The subunit arrangement of Chlamydomonas rubisco is the same as those of the previously solved form I rubiscos. Especially, the present structure is very similar to the activated spinach structure complexed with 2-CABP in the L-subunit folding and active-site conformation, but differs in S-subunit folding. The central insertion of the Chlamydomonas S-subunit forms the longer βA-βB loop that protrudes deeper into the solvent channel of rubisco than higher plant, cyanobacterial, and red algal (red-like) βA-βB loops. The C-terminal extension of the Chlamydomonas S-subunit does not protrude into the solvent channel, unlike that of the red algal S-subunit, but lies on the protein surface anchored by interactions with the N-terminal region of the S-subunit. Further, the present high-resolution structure has revealed novel post-translational modifications. Residue 1 of the S-subunit is Nα-methylmethionine, residues 104 and 151 of the L-subunit are 4-hydroxyproline, and residues 256 and 369 of the L-subunit are Sγ-methylcysteine. Furthermore, the unusual electron density of residue 471 of the L-subunit, which has been deduced to be threonine from the genomic DNA sequence, suggests that the residue is isoleucine produced by RNA editing or Oγ-methylthreonine.

Parasitic protozoa Trypanosoma causes diseases in both human and domestic animals.In human, the disease is characterized by clinical symptoms such as fever, headache, body pain, sleepiness, and immunosuppression.Bioactive substances produced during the infection cause these symptoms, whose molecular mechanisms remain to be elucidated.Prostaglandins (PGs) of 2series are metabolites of arachidonic acid formed by the oxidative cyclization of the central five carbon atoms within this fatty acid.It is well documented that PGD2 is one of the endogenous sleep-promoting substances.PGD2 and PGE2 play important roles in clinical and pathological manifestations (such as somnolence, immunosupression acute fever, shivering, headache) in mammals.Also, it is often believed that host cells solely produce prostaglandins, which mediate inflammatory responses during infection with parasitic protozoa.Trypanosoma produces PGD2, PGE2, and PGF2α (PGF2α > PGE2 > PGD2), and releases them in the medium and out of parasitized red blood cells.We crystallized the NADP-dependent prostaglandin F2α synthase from T. brucei used by the hanging-drop vapour-diffusion method.The crystal was tetragonal system, space group P41212, with unit-cell parameters a = b = 112.3,c = 140.0Å.The X-ray diffraction data were collected from a crystal to a resolution of 2.8 Å using our home facility at room temperature.

CRYSTAL STRUCTURE OF GALDIERIA RUBISCO REVEALS A NOVEL CLOSURE MECHANISM OF THE ACTIVE SITE E. Mizohata Y. Okano Y. Xie H. Matsumura H. Sugawara 4 T. Inoue A. Yokota Y. Kai Osaka University Dept. of Materials Chem., Gradutate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita OSAKA 565-0871 JAPAN Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5, Takayama, Ikoma, Nara 630-0101, Japan Pl t Molecular Physiology Laboratory, Research Institute of Innovative Technology for the Earth (RITE), 9-2 Kizugawadai, Kizu-cho, Soraku-gun, Kyoto 6190292, Japan Metabolic Function Research Group, RIKEN Plant Science Center, Hirosawa 2-1, Wako, Saitama 351-0198, Japan

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the reactions of carboxylation and oxygenation of ribulose‐1,5‐bisphosphate. These reactions require that the active site should be closed by a flexible loop (loop 6) of the large subunit. Rubisco from a red alga, Galdieria partita, has the highest specificity for carboxylation reaction among the Rubiscos hitherto reported. The crystal structure of unactivated Galdieria Rubisco has been determined at 2.6 Å resolution. The electron density map reveals that a sulfate binds only to the P1 anion‐binding site of the active site and the loop 6 is closed. Galdieria Rubisco has a unique hydrogen bond between the main chain oxygen of Val332 on the loop 6 and the ϵ‐amino group of Gln386 of the same large subunit. This interaction is likely to be crucial to understanding for stabilizing the loop 6 in the closed state and to making a higher affinity for anionic ligands.

Prostaglandin F(2 alpha) is a potent mediator of various physiological and pathological processes. Trypanosoma brucei prostaglandin F(2 alpha) synthase (TbPGFS) catalyzes the NADPH-dependent reduction of 9,11-endoperoxide PGH(2) to PGF(2 alpha), and could thus be involved in the elevation of the PGF(2 alpha) concentration during African trypanosomiasis. In the present report, the purification and crystallization of recombinant TbPGFS are described. The active recombinant enzyme was crystallized by the hanging-drop vapor-diffusion meth-od using ammonium sulfate as a precipitant. The crystal belonged to a tetragonal space group, P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters of a = b = 112.3 A, and c = 140.0 A. Native data up to 2.6 A resolution were collected from the crystal using our home facility.