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J. Biol. Chem., Vol. 280, Issue 28, 26371-26382, July 15, 2005

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Structural and Mutational Analysis of Trypanosoma brucei Prostaglandin H₂ Reductase Provides Insight into the Catalytic Mechanism of Aldo-ketoreductases^*

From the ^aDepartment of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan, Departments of ^eMaterials Chemistry and ^gMolecular Protozoology, Osaka University, Yamada-oka, Suita, Osaka 565-0871, Japan, ^bUnited States Army Medical Research Unit-Kenya, Unit 64109, APO AE 09831-64109, ⁱPhysiologisch-chemisches Institut der Universität Tübingen, Hoppe-Seyler-Strasse 4, 72076 Tübingen, Germany, ^jMARUWA Foods Industries Inc., 170 Tsutsui-cho, Yamatokoriyama, Nara 639-1123, Japan, ^kDepartment of Food and Nutrition, Tsu City College, 157 Ishinden, Tsu City, Mie 514-0112, Japan, and ^fStructure and Function of Biomolecules Group, PRESTO, Japan Science Technology Agency, Honcho Kawaguchi, Saitama 332-0012, Japan

Trypanosoma brucei prostaglandin F₂ synthase is an aldo-ketoreductase that catalyzes the reduction of prostaglandin H₂ to PGF₂ 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 (/)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.

Received for publication, December 9, 2004 , and in revised form, April 19, 2005.

The atomic coordinates and structure factors (code 1VBJ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Grants-in-aid for Scientific Research (B) 14370087 (to K. B. K. and T. I.), 14021130 (to K. B. K.), and 16017260 (to T. I.) from the Ministry of Education, Culture, Sport, Science and Technology, Japan; grants from PRESTO Project, Japan Science and Technology Agency (to T. I.); grants from Ono Medical Research Foundation (to K. B. K.); grants from "Applied Research Pilot Project for the Industrial Use of Space" promoted by Japan Aerospace Exploration Agency and Japan Space Utilization Promotion Center (to Y. U.); grants from Handai Frontier Research Center (to T. I.); and by Japan Society for the Promotion of Science Postdoctoral Fellowship 02271 (to Z. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^c These authors contributed equally to this work.

^h Present address: Dept. of Neurology, Beth Israel Deaconess Medical Center, Harvard University, 77 Ave. Louis Pasteur, Boston, MA 02115.

^d To whom correspondence should be addressed: United States Army Medical Research Unit-Kenya, Unit 64109, APO AE 09831-64109. Tel.: 254-733-665210; Fax: 1-202-478-5084; E-mail: bkubata{at}nairobi.mimcom.net.

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