Human prostaglandin reductase 1 (PTGR1): Substrate specificity, site-directed mutagenesis and catalytic mechanism

The work here presented is part of the structural and functional characterization of the protein superfamily of medium-chain dehydrogenases/reductases (MDRs). The thesis focuses on prostaglandin reductases (PTGRs), a group of proteins belonging to the alkenal/one oxidoreductase (AOR) protein family. The physiological substrates of PTGRs, prostaglandins (PGs), are lipid compounds derived from arachidonic acid by the action of cyclooxygenases, acting locally as messenger molecules in a wide variety of physiological processes. In the inactivating pathway of PGs, the first metabolic intermediates are 15-keto-PGs, which are further converted into 13,14-dihydro-15-keto-PGs by various enzymes having 15-keto-PG reductase activity. Three human PTGRs perform the first irreversible step of the degradation pathway. The first chapter deals with the characterization of the recombinant human PTGR1. Only a partial characterization of this enzyme, isolated from human placenta, had been previously reported. In the present work, we have performed an extensive kinetic characterization of PTGR1, which catalyzes the NADPH-dependent reduction of the α,β-double bond of aliphatic and aromatic aldehydes and ketones, and of 15-keto-PGs. PTGR1 also shows low activity in the oxidation of leukotriene B4. The best substrates in terms of kcat/Km were 15 keto-PGE2, trans-3-nonen-2-one and trans-2-decenal. Molecular docking simulations, based on the three-dimensional structure of the human enzyme (PDB ID 2Y05), and site-directed mutagenesis studies were performed to pinpoint important structural determinants, highlighting the role of Arg56 and Tyr245. Finally, inhibition analysis was done using non-steroidal anti-inflammatory drugs (NSAIDs) as potential inhibitors. The second chapter is centered on the activity of PTGR1 with 15d-PGJ2, which is a natural ligand of peroxisome proliferator-activated receptor gamma and a highly reactive electrophilic molecule. Inactivation of 15d-PGJ2 can be achieved by double-bond hydrogenation catalyzed by AORs, such as PTGR1. By using 1H and 13C NMR spectroscopy, we have identified two different diastereoisomers, (12R)- and (12S)-12,13-dihydro-15d-PGJ2, as the products of human PTGR1 activity, proving the enzymatic hydrogenation of the C12-C13 α,β-double bond. Molecular docking of 15d PGJ2 into the crystallographic structure of human PTGR1 predicts three tyrosine residues (Tyr49, Tyr245 and Tyr273) making van der Waals interactions with the substrate and a properly positioned Cβ atom for hydride transfer from the C4 atom of the NADPH nicotinamide. However, Tyr49 is not well oriented while substitution of either Tyr245 or Tyr273 by site-directed mutagenesis yields active enzyme, thus discarding their participation as proton donor residues. Considering these results, we propose a catalytic mechanism where the NADPH hydride first attacks the electrophilic C13 atom, and then protonation of the C12 atom takes place with little stereoselectivity. Since a suitably positioned proton donor residue is not available, a solvent proton could be added from both substrate faces, leading to the formation of the two diastereomeric products. The here described mechanism is also fully compatible with the hydrogenation of the C13-C14 double bond of 15-keto-PGs by PTGR1.