Structural and Functional Analyses of Wild Type and Mutant Prostacyclin Synthase

• Main Authors: Yi-Ching Li, 李宜靜
• Other Authors: 詹迺立
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/38692489937687741657

博士 === 國立中興大學 === 生物化學研究所 === 99 === Prostacyclin synthase (PGIS) plays crucial roles in cardiovascular function by catalyzing an isomerization of prostaglandin H2 (PGH2) to produce prostacyclin, a key bioactive prostanoid known for its potent vasodilation and anti-platelet aggregation activities. PGIS belongs to the heme-contaning cytochrome P450 enzyme superfamily that participates in various crucial biological oxidation reactions. PGIS is classified as the class III P450 and is unique among the P450 family members, it catalyzes a sterospecific isomerizaton reaction and requires neither O2 nor any external reductase for function. To understand the unique characteristics and structural basis for this stereospecific catalysis, we have determined the crystal structures of the ligand-free and PGH2 analog U51605-bound PGIS. These structures demonstrate a stereospecific substrate binding and suggested features of the enzyme that may 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. Further, our results indicate that these conformational changes are ligand-specific. The U51605-bound structure also provides a view of the substrate entrance and product exit channels. Structural comparison has led to the identification of residues in the active site that may be crucial for catalysis. In particular, we noticed that the side chain of N277 appears positioned to form hydrogen-bonding interaction with the C-9 oxygen of substrate PGH2, likely contributing directly to the cleavage of endoperoxide bond. The position of W272 is also notable among the hydrophobic residues in the active site. With its indole side chain lies parallel to the heme, we originally suspected that W272 might serve as a ceiling in the active site to constrain the spatial position of substrate. As expected, mutations of these two residues impair the catalytic function of PGIS. To understand the functional perturbations of these mutations in greater detail, we have determined the crystal structures of the ligand-free and PGH2 analog U51605-bound forms of the N277A and W272A mutant PGIS. This work establishes that the polar (H-bonding) interaction between N277 side chain and the endoperoxide moiety of the substrate PGH2 contribute directly to catalysis, and that W272 is required for optimal catalytic function by positioning, via a structured network of water molecules in the active site, the N277 side chain at a location suitable for making H-bonding with substrate PGH2. Moreover, because the bound conformations of U51605 in both W272A and N277A mutants are the same as the wild-type enzyme, therefore, we conclude that the roles played by these two residues are largely catalytic. The substrate binding geometry is probably maintained by its interactions with the heme group.