RESUMEN
Prostacyclin synthase (CYP8A1) is an enzyme responsible for the biosynthesis of prostacyclin (PGI2) which inhibits platelet activation and exhibits anti-inflammatory effect. The objectives of this study were to identify CYP8A1 genetic variants and characterize functional consequences of CYP8A1 variants. In total, 27 variants including four previously unidentified single-nucleotide polymorphisms (SNPs) were identified by direct DNA sequencing in Koreans (n=48). Among them, CYP8A1 A447T and E314Stop were newly assigned as CYP8A1(∗)5 and CYP8A1(∗)6 by the Human Cytochrome P450 Allele Nomenclature Committee, respectively. CYP8A1(∗)5 was found in the heme binding area in three individuals as a heterozygous mutation. To investigate the functional change of CYP8A1(∗)5, CYP8A1(∗)5 and wild-type CYP8A1 protein were overexpressed in an Escherichia coli expression system and purified. Metabolism of PGH2 by the CYP8A1(∗)5 protein exhibited significantly decreased activity, resulting in a 45% decrease in Vmax and a 1.8-fold decrease in intrinsic clearance compared to the wild-type. Based on the predicted crystal structure of CYP8A1(∗)5 using the Molecular Operating Environment platform, the distance from CYP8A1 Cys441 to the heme was altered with a significantly changed binding free energy for the mutant protein. Further studies would be needed to determine the effect of CYP8A1(∗)5 on PGI2 levels in humans.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Variación Genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Codón sin Sentido , Simulación por Computador , Sistema Enzimático del Citocromo P-450/química , ADN/genética , Hemo/metabolismo , Heterocigoto , Humanos , Cinética , Desequilibrio de Ligamiento , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Polimorfismo de Nucleótido Simple , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , República de Corea , Homología de Secuencia de AminoácidoRESUMEN
Biosensor experiments on investigation of interaction between prostacyclin synthase (PGIS) and different proteins of the cytochrome P450 monooxygenase systems were perfomed. Interaction of PGIS with microsomal (CYP21A2, CYP2E1) and mitochondrial (CYP27A1, CYP11B1, CYP11B2, CYP11A1) cytochrome P450s was detected. Kinetic and equilibrium parameters of protein complexes formation were determined. Data obtained suggest an essential role of these hemoproteins interaction in regulation of prostacyclin and thromboxane A2 biosynthesis.