Structural and mutational analysis of Trypanosoma brucei prostaglandin H2 reductase provides insight into the catalytic mechanism of aldo-ketoreductases.
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.
Kilunga
Kubata Bruno
KB
Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan. bkubata@nairobi.mimcom.net
Inoue
Tsuyoshi
T
Okano
Yousuke
Y
Kabututu
Zakayi
Z
Martin
Samuel K
SK
Lazarus
Michael
M
Duszenko
Michael
M
Sumii
Yuichi
Y
Kusakari
Yukiko
Y
Matsumura
Hiroyoshi
H
Kai
Yasushi
Y
Sugiyama
Shigeru
S
Inaka
Kouji
K
Inui
Takashi
T
Urade
Yoshihiro
Y
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D002951:Citrates / Q000737:chemistry; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D019143:Evolution, Molecular; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006911:Hydroxyprostaglandin Dehydrogenases / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism*; D044262:Prostaglandin H2 / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011522:Protons; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014346:Trypanosoma brucei brucei / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry; D014466:Ultraviolet Rays
D002951:Citrates; D018076:DNA, Complementary; D008024:Ligands; D011522:Protons; D044262:Prostaglandin H2; D014443:Tyrosine; D010088:Oxidoreductases; D006911:Hydroxyprostaglandin Dehydrogenases; C031051:prostaglandin-F synthase