Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

Abstract

The possible active site Cys 441 in the Cys-pocket and Glu 347 and Arg ~s° of the EXXR motif of the human prostacyclin synthase, which catalyzes the conversion of prostaglandin H2 to prostacyclin, were subjected to site-directed mutagenesis in order to understand the role of these residues in expressing the enzymatic activity. Five expression vectors encoding the mutant enzymes with a single replacement, Cys441Ala, Cys441Ser, Cys441His, Glu347Ala and Arg3S°Ala, as well as the wild-type enzyme were expressed in 293 cells. The microsomal fraction of the cells expressing the wild-type enzyme showed a specific activity of 96 nmol 6-keto-PGF1Jmin per mg protein. All of the mutant enzymes examined showed no detectable enzyme activity, although immunoblot analysis demonstrated that levels of all the expressed mutant enzymes were similar to that of the wild-type enzyme. These results indicated that the Cys 441 in the Cyspocket, and Glu 347 and Arg 35° of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.

Prostacyclin-dependent Apoptosis Mediated by PPARδ

“…An expression vector encoding human PGIS (29) or control vector was transfected into HEK-293 cells, and cell viability was determined. As shown in Fig.…”

“…Cell Culture and Transfection-HEK-293 (29), CV-1, and bovine aortic endothelial cells (BAEC) (30) (1.2 ϫ 10 4 cells/cm 2 ) were cultured in medium A. Caco-2 cells (1.0 ϫ 10 5 cells/30-mm dish coated with collagen) were cultured in RPMI 1640 supplemented with 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin. For expression of PGISwt, PGISC441A, COX-1, and COX-2, plasmid vectors pCMV/ PGISWT (29), pCMV/PGISC441A (29), pcDNACOX-1 (31), and pcDNACOX-2 (31) were used, respectively.…”

“…For expression of PGISwt, PGISC441A, COX-1, and COX-2, plasmid vectors pCMV/ PGISWT (29), pCMV/PGISC441A (29), pcDNACOX-1 (31), and pcDNACOX-2 (31) were used, respectively. HEK-293, Caco-2 and CV-1 cells were transfected with DNA using LipofectAMINE.…”

Three-dimensional Structure of Steroid 21-Hydroxylase (Cytochrome P450 21A2) with Two Substrates Reveals Locations of Disease-associated Variants

“…The Glu and Arg and residues from the meander region can form a set of salt bridge/hydrogen bonding interactions that participate in the formation of the tertiary structure. There is evidence that suggests that EXXR is linked somehow to heme association with the P450 polypeptide (30,37). In CYP21A2, Glu-350 and Arg-353 of the EXXR motif show standard ion pair interaction and also make hydrogen bonding interactions with residues Pro-400, Glu-402, Arg-404, Thr-347, and His-391 from the meander region.…”

The cytochrome P450 gene family CYP157 does not contain EXXR in the K‐helix reducing the absolute conserved P450 residues to a single cysteine

“…The several previous studies indicating that EXXR is critical for correct P450 folding include mutagenesis of Arg365 in CYP19A1 which led to the production of inactive and misfolded protein [17] and mutagenesis of the corresponding Arg residue in CYP1A2 which led to inactive protein that could not bind haem [10]. In other studies, Hatae and colleagues [9] mutated both Glu347 and Arg350 of CYP8A1 to Ala which again abolished enzyme activity while mutation of Glu359 and Arg362 in bovine CYP17A1 also led to inactive protein (Barnes and Waterman, unpublished data). In all cases, no correctly folded P450s were observed.…”