Prostacyclin synthase active sites. Identification by molecular modeling-guided site-directed mutagenesis.
Prostacyclin synthase (PGIS), a cytochrome P450 enzyme, catalyzes the biosynthesis of a physiologically important molecule, prostacyclin. In this study we have used a molecular modeling-guided site-directed mutagenesis to predict the active sites in substrate binding pocket and heme environment of PGIS. A three-dimensional model of PGIS was constructed using P450BM-3 crystal structure as the template. Our results indicate that residues Ile67, Val76, Leu384, Pro355, Glu360, and Asp364, which were suggested to be located at one side of lining of the substrate binding pocket, are essential for catalytic activity. This region containing beta1-1, beta1-2, beta1-3, and beta1-4 strands is predicted well by the model. At the heme region, Cys441 was confirmed to be the proximal axial ligand of heme iron. The conserved Phe and Arg in P450BM-3 were substituted by Leu112 and Asp439, respectively in PGIS. Alteration of Leu112 to Phe retained the activity, indicating that Leu112 is a functional substitution for Phe. In contrast, mutant Asp439 --> Ala exhibited a slight increase in activity. This result implies a difference in the heme region between P450BM-3 and PGIS. Our results also indicate that stability of PGIS expression is not affected by heme site or active site mutations.
Shyue
S K
SK
Vascular Biology Research Center and Division of Hematology, University of Texas Medical School, Houston, Texas 77030, USA.
Ruan
K H
KH
Wang
L H
LH
Wu
K K
KK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002855:Chromatography, Thin Layer; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment
D003577:Cytochrome P-450 Enzyme System; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase