Structures of Prostacyclin Synthase and Its Complexes with Substrate Analog and Inhibitor Reveal a Ligand-specific Heme Conformation Change

Yi-Ching Li,Chia-Wang Chiang,Hui-Chun Yeh,Pei-Yung Hsu,Frank G Whitby,Lee-Ho Wang,Nei-Li Chan

doi:10.1074/jbc.m707470200

Abstract

Prostacyclin synthase (PGIS) is a cytochrome P450 (P450) enzyme that catalyzes production of prostacyclin from prostaglandin H(2). PGIS is unusual in that it catalyzes an isomerization rather than a monooxygenation, which is typical of P450 enzymes. To understand the structural basis for prostacyclin biosynthesis in greater detail, we have determined the crystal structures of ligand-free, inhibitor (minoxidil)-bound and substrate analog U51605-bound PGIS. These structures demonstrate a stereo-specific substrate binding and suggest features of the enzyme that facilitate isomerization. Unlike most microsomal P450s, where large substrate-induced conformational changes take place at the distal side of the heme, conformational changes in PGIS are observed at the proximal side and in the heme itself. The conserved and extensive heme propionate-protein interactions seen in all other P450s, which are largely absent in the ligand-free PGIS, are recovered upon U51605 binding accompanied by water exclusion from the active site. In contrast, when minoxidil binds, the propionate-protein interactions are not recovered and water molecules are largely retained. These findings suggest that PGIS represents a divergent evolution of the P450 family, in which a heme barrier has evolved to ensure strict binding specificity for prostaglandin H(2), leading to a radical-mediated isomerization with high product fidelity. The U51605-bound structure also provides a view of the substrate entrance and product exit channels.

Highlights

Prostacyclin ( known as prostaglandin I2 (PGI2))4 is a potent inhibitor of vasoconstriction, platelet activation, and aggregation [1]
Asn419, one sits at the active site (Fig. 4A), one sits in the middle of the equivalent to the conserved Arg/His, brings about the most helices AЈ, BЈ, and FЈ, and strands ␤1–3 and ␤1– 4 that is prominent conformational change within the cysteine ligand the substrate entrance channel we previously proposed for loop, with its side chain rotating by ϳ90° toward the heme
Five water molecules in Channel 1 are present in the ligand-free structure, whereas only one is found in the U51605-bound structure. These findings indicate that a large number of water molecules originally seen in the active site and substrate entrance channel of the ligand-free protein are excluded upon U51605 binding, which greatly reduces the polarity of these regions

Summary

Introduction

Prostacyclin ( known as prostaglandin I2 (PGI2))4 is a potent inhibitor of vasoconstriction, platelet activation, and aggregation [1]. The hPGIS is 10 amino acid residues longer at both the F helix and the J/K loop than zPGIS, both regions are located on the protein surface, and only small and localized differences were observed due to this sequence divergence (Supplemental Fig. 2B).

Results

Conclusion