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

Kubata Bruno Kilunga,Tsuyoshi Inoue,Yousuke Okano,Zakayi Kabututu,Samuel K Martin,Michael Lazarus,Michael Duszenko,Yuichi Sumii,Yukiko Kusakari,Hiroyoshi Matsumura,Yasushi Kai,Shigeru Sugiyama,Kouji Inaka,Takashi Inui,Yoshihiro Urade

doi:10.1074/jbc.m413884200

Abstract

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.

Highlights

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
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
We show that Tyr52 acts as the general acid catalyst for 9,10-PQ reduction, and we elucidate the catalytic mechanism of a PGH2 reductase and provide an insight into the catalytic specificity of AKRs

Summary

Structural and Mutational Analysis of Trypanosoma brucei

Prostaglandin H2 Reductase Provides Insight into the Catalytic Mechanism of Aldo-ketoreductases*. Earlier structural and functional studies on AKRs have elucidated the catalytic mechanism of the oxidation/reduction reaction for physiological substrates such as monosaccharides (8 –10), steroid hormones [4], and aldehydes [1, 11]. These studies revealed that bacterial and mammalian AKRs catalyze the oxidation/reduction through a catalytic mechanism that involves a catalytic tetrad of aspartate, tyrosine, lysine, and histidine. We propose a catalytic mechanism by which a catalytic dyad of Lys and His110 in TbPGFS is involved in proton transfer to the 9,11-endoperoxide moiety of PGH2

MATERIALS AND METHODS

Root mean square deviations from ideal geometry

RESULTS AND DISCUSSION

The presence of short loops in TbPGFS and Corynebacterium

The loss of activity observed throughout this study could

Substrate pKa