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Background: Cytochrome P450 17A1 (CYP17A1) is a key steroidogenic enzyme that catalyzes both 17α-hydroxylase and 17,20-lyase reactions, thereby regulating glucocorticoid and sex steroid biosynthesis. Cytochrome b5 (b5) is known to selectively enhance the 17,20-lyase activity of CYP17A1; however, the molecular mechanism underlying this modulation remains incompletely understood.
Methods: Recombinant human CYP17A1, rat NADPH–P450 reductase (POR), and human cytochrome b5 (wild-type and mutants E48A and E48K/E49K) were heterologously expressed in Escherichia coli and purified to homogeneity. Catalytic activities were evaluated using a reconstituted system consisting of CYP17A1, POR, and varying ratios of b5. Steroid products were quantified by UPLC–MS/MS analysis. Steady-state kinetic parameters were determined to compare the effects of wild-type and mutant b5 proteins.
Results: CYP17A1 activity was maximized at a CYP17A1:b5 molar ratio of 1:5, whereas excessive b5 led to a significant reduction in overall catalytic activity. While hydroxylase activity showed comparable catalytic efficiency between wild-type and mutant b5, lyase activity was markedly reduced in the presence of E48A and E48K/E49K variants. Notably, at the optimal molar ratio (1:5), the E48K/E49K mutant exhibited approximately 37% lower catalytic efficiency for the lyase reaction compared with wild-type b5.
Conclusions: These findings indicate that residues E48 and E49 of cytochrome b5 play a critical role in functional interaction with CYP17A1. Furthermore, the data support a model in which b5 acts as an allosteric modulator of CYP17A1, while excessive b5 may competitively interfere with POR binding and disrupt efficient electron transfer. Based on these observations, a competitive interaction model for the CYP17A1–b5–POR ternary complex is proposed. This study provides mechanistic insight into the regulation of CYP17A1 and contributes to the rational design of selective modulators targeting steroidogenic pathways.
Acknowledgments: Supported by the National Research Foundation of Korea (NRF) grant RS-2024-00335140
