Two surfaces of cytochrome b₅ with major and minor contributions to CYP3A4-catalyzed steroid and nifedipine oxygenation chemistries

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• 作者：Hwei-Ming Peng ; Richard J. Auchus
• 关键词：Cytochrome b5 ; Testosterone ; CYP3A4 ; Allostery ; Cytochrome P450 ; Drug oxidation
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：1 January, 2014
• 年：2014
• 卷：541
• 期：Complete
• 页码：53-60
• 全文大小：1502 K

文摘

Conserved human cytochrome b₅ (b₅) residues D58 and D65 are critical for interactions with CYP2E1 and CYP2C19, whereas E48 and E49 are essential for stimulating the 17,20-lyase activity of CYP17A1. Here, we show that b₅ mutations E48G, E49G, D58G, and D65G have reduced capacity to stimulate CYP3A4-catalyzed progesterone and testosterone 6尾-hydroxylation or nifedipine oxidation. The b₅ double mutation D58G/D65G fails to stimulate these reactions, similar to CYP2E1 and CYP2C19, whereas mutation E48G/E49G retains 23-42% of wild-type stimulation. Neither mutation impairs the activity stimulation of wild-type b₅, nor does mutation D58G/D65G impair the partial stimulation of mutations E48G or E48G/E49G. For assays reconstituted with a single phospholipid, phosphatidyl serine afforded the highest testosterone 6尾-hydroxylase activity with wild-type b₅ but the poorest activity with b₅ mutation E48G/E49G, and the activity stimulation of mutation E48G/E49G was lost at [NaCl] > 50 mM. Cross-linking of CYP3A4 and b₅ decreased in the order wild-type > E48G/E49G > D58G/D65G and varied with phospholipid. We conclude that two b₅ acidic surfaces, primarily the domain including residues D58-D65, participate in the stimulation of CYP3A4 activities. Our data suggest that a minor population of CYP3A4 molecules remains sensitive to b₅ mutation E48G/E49G, consistent with phospholipid-dependent conformational heterogeneity of CYP3A4.