| First Author | Lee SG | Year | 2023 |
| Journal | J Inorg Biochem | Volume | 240 |
| Pages | 112085 | PubMed ID | 36640554 |
| Mgi Jnum | J:338155 | Mgi Id | MGI:7510298 |
| Doi | 10.1016/j.jinorgbio.2022.112085 | Citation | Lee SG, et al. (2023) Hydroxylation and lyase reactions of steroids catalyzed by mouse cytochrome P450 17A1 (Cyp17a1). J Inorg Biochem 240:112085 |
| abstractText | Cytochrome P450 17A1 (CYP17A1) catalyzes 17alpha-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni(2+)-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17alpha-OH progesterone, pregnenolone, and 17alpha-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17alpha-hydroxylation and 17,20-lyase reactions were studied using ultra-performance liquid chromatography (UPLC)-mass spectrometry analysis. Mouse Cyp17a1 showed cytochrome b(5) stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (k(cat)/K(m)) were observed with mouse Cyp17a1. In the reactions of Delta4-steroids (progesterone and 17alpha-OH progesterone), the specificity constants were 2100 times higher than the human enzyme. The addition of cytochrome b(5) produced significant stimulation of 17,20-lyase activities of mouse Cyp17a1. Two Arg mutants of mouse Cyp17a1 (R347H and R358Q) displayed a larger decrease in 17,20-lyase reaction (from 17alpha-OH pregnenolone to dehydroepiandrosterone, DHEA) than 17alpha-hydroxylation, indicating that -as in human CYP17A1-these basic residues in mouse Cyp17a1 are important in interacting with the cytochrome b(5) protein in the lyase reactions. |
