Molecular insights into CYP19A1 mutations and their role in estrogen production

Human cytochrome P450 enzyme CYP19A1, commonly referred to as aromatase, plays a critical role in estrogen biosynthesis by catalyzing the conversion of C19 androgens into aromatic C18 estrogens. Alterations in aromatase activity have been implicated in the development and progression of estrogen-dependent diseases. Genetic mutations, including nonsynonymous single nucleotide polymorphisms (SNPs), can markedly impact the catalytic function of CYP19A1. In this study, we investigated the functional implications of six CYP19A1 variants (R192H, R192Q, T201M, R264C, P308F, and M364T). These variants were generated via site-directed mutagenesis, expressed in Escherichia coli, and purified. The T201M, R264C, and P308F variants exhibited measurable expression levels ranging from 90 to 150nmol P450 per liter culture, whereas no detectable P450 holoenzyme was observed for the R192H, R192Q, and M364T variants. Spectral binding assays revealed typical type I spectral shifts upon androgen binding for all purified variants with tight affinity. Catalytic activities of testosterone and androstenedione aromatization were assessed by UPLC-mass spectrometry, and steady-state kinetic analyses demonstrated that T201M and R264C retained catalytic efficiencies comparable to wild-type, with efficiency ratios of 1.1-0.9 for androstenedione and 0.6-0.8 for testosterone, respectively, relative to the wild-type. In contrast, P308F showed marked reductions in catalytic efficiencies (0.2 and 0.1), driven by decreases in both k_cat and K_m values. Structural analysis indicates that the proline residue at position 308, situated within the I-helix, is likely critical for stabilizing a catalytically active conformation of the enzyme-substrate complex. These results provide valuable insights into the functional role of CYP19A1 in estrogen biosynthesis and could inform the design of innovative therapies for estrogen-related diseases.