Structural basis of leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase catalytic mechanism and a possible Src homology 3 domain binding loop

Abstract

The bifunctional leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB4 12-HD/ PGR) is an essential enzyme for eicosanoid inactivation. It is involved in the metabolism of the E and F series of 15-oxo-prostaglandins (15-oxo-PGs), leukotriene B4 (LTB4), and 15-oxo-lipoxin A4 (15-oxo-LXA4). Some nonsteroidal anti-inflammatory drugs (NSAIDs), which primarily act as cyclooxygenase inhibitors also inhibit LTB4 12-HD/PGR activity. Here we report the crystal structure of the LTB4 12-HD/PGR, the binary complex structure with NADP+, and the ternary complex structure with NADP+ and 15-oxo-PGE2. In the ternary complex, both in the crystalline form and in solution, the enolate anion intermediate accumulates as a brown chromophore. PGE2 contains two chains, but only the ω-chain of 15-oxo-PGE2 was defined in the electron density map in the ternary complex structure. The ω-chain was identified at the hydrophobic pore on the dimer interface. The structure showed that the 15-oxo group forms hydrogen bonds with the 2′-hydroxyl group of nicotine amide ribose of NADP+ and a bound water molecule to stabilize the enolate intermediate during the reductase reaction. The electron-deficient C13 atom of the conjugated enolate may be directly attacked by a hydride from the NADPH nicotine amide in a stereospecific manner. The moderate recognition of 15-oxo-PGE2 is consistent with a broad substrate specificity of LTB4 12-HD/PGR. The structure also implies that a Src homology domain 3 may interact with the left-handed proline-rich helix at the dimer interface and regulate LTB4 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the ω-chain.