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Abstract
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.
Highlights
Prostacyclin synthase (PGIS), a cytochrome P450 enzyme, catalyzes the biosynthesis of a physiologically important molecule, prostacyclin
Since arachidonic acid (AA) is a functional substrate of P450BM-3, and the substrate is closely related to the substrate of thromboxane A2 synthase (TXAS) and PGI2 synthase (PGIS), we reasoned that the crystal structure of the hemoprotein domain could serve as a useful template for constructing a three-dimensional model of these two enzymes
Substrate Binding Pocket—A three-dimensional model based on the crystal structure of the hemoprotein domain of P450BM-3 was constructed
Summary
Prostacyclin synthase (PGIS), a cytochrome P450 enzyme, catalyzes the biosynthesis of a physiologically important molecule, prostacyclin. On sequence comparison with P450s, we observed the conservation of several P450 structural elements in PGIS, including a putative membrane-anchoring segment, a helix I which forms an ␣-helix backbone through the center of the enzyme, and a heme binding pocket. This raised the possibility of mapping the active site amino acid residues by molecular modeling-guided site-directed mutagenesis. We constructed a PGIS three-dimensional model based on the crystallographic structure of P450BM-3 From this model, we identified several amino residues potentially involved in substrate access and binding and in heme binding. Results from these experiments have allowed for identifying amino acid residues in the substrate channel that are important in substrate access and binding and in interaction with heme
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