Structural and mutational analysis of Trypanosoma brucei prostaglandin H2 reductase provides insight into the catalytic mechanism of aldo-ketoreductases

Trypanosoma brucei prostaglandin F-2 alpha synthase is an aldo-ketoreductase that catalyzes the reduction of prostaglandin H-2 to PGF(2 alpha) in addition to that of 9,10- phenanthrenequinone. We report the crystal structure of TbPGFS.NADP(+).citrate at 2.1 angstrom 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 Lys(77) and His(110), and a triad of Tyr(52), Lys(77), and His(110) are essential for the reduction of PGH(2) and 9,10- PQ, respectively. Structural and kinetic analysis revealed that His(110), acts as the general acid catalyst for PGH(2) reduction and that Lys(77) facilitates His(110) protonation through a water molecule, while exerting an electrostatic repulsion against His(110) that maintains the spatial arrangement which allows the formation of a hydrogen bond between His(110) and C-11 carbonyl ofPGH(2). We also show that Tyr(52) acts as the general acid catalyst for 9,10- PQ reduction, and thus we not only elucidate the catalytic mechanism of a PGH(2) reductase but also provide an insight into the catalytic specificity of AKRs.