TY - JOUR
T1 - Spatially restricted substrate-binding site of cortisol-synthesizing CYP11B1 limits multiple hydroxylations and hinders aldosterone synthesis
AU - Mukai, Kuniaki
AU - Sugimoto, Hiroshi
AU - Kamiya, Katsumasa
AU - Suzuki, Reiko
AU - Matsuura, Tomomi
AU - Hishiki, Takako
AU - Shimada, Hideo
AU - Shiro, Yoshitsugu
AU - Suematsu, Makoto
AU - Kagawa, Norio
N1 - Funding Information:
We thank Dr. Yasuhiro Sagara for kindly providing a cDNA encoding bovine adrenodoxin and Dr. Fumiko Mitani for generously providing purified bovine adrenodoxin reductase. This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science ( 26461387 and 17K09890 to K.M. and 20K03794 to K.K.) and by Keio Gijuku Academic Development Funds. Infrastructure of LC-MS/MS was supported by JST ERATO Suematsu Gas Biology from the Japan Science and Technology Agency (to M.S.).
Publisher Copyright:
© 2021 The Authors
PY - 2021/1
Y1 - 2021/1
N2 - Human cytochromes P45011β (CYP11B1) and P450aldo (CYP11B2) are monooxygenases that synthesize cortisol through steroid 11β-hydroxylation and aldosterone through a three-step process comprising 11β-hydroxylation and two 18-hydroxylations, respectively. CYP11B1 also catalyzes 18-monohydroxylation and 11β,18-dihydroxylation. To study the molecular basis of such catalytic divergence of the two enzymes, we examined a CYP11B1 mutant (Mt-CYP11B1) with amino acid replacements on the distal surface by determining the catalytic activities and crystal structure in the metyrapone-bound form at 1.4-Å resolution. Mt-CY11B1 retained both 11β-hydroxylase and 18-hydroxylase activities of the wild type (Wt-CYP11B1) but lacked 11β,18-dihydroxylase activity. Comparisons of the crystal structure of Mt-CYP11B1 to those of Wt-CYP11B1 and CYP11B2 that were already reported show that the mutation reduced the innermost space putatively surrounding the C3 side of substrate 11-deoxycorticosterone (DOC) bound to Wt-CYP11B1, while the corresponding space in CYP11B2 is enlarged markedly and accessible to bulk water through a channel. Molecular dynamics simulations of their DOC-bound forms supported the above findings and revealed that the enlarged space of CYP11B2 had a hydrogen bonding network involving water molecules that position DOC. Thus, upon positioning 11β-hydroxysteroid for 18-hydroxylation in their substrate-binding sites, steric hindrance could occur more strongly in Mt-CYP11B1 than in Wt-CYP11B1 but less in CYP11B2. Our investigation employing Mt-CYP11B1 sheds light on the divergence in structure and function between CYP11B1 and CYP11B2 and suggests that CYP11B1 with spatially-restricted substrate-binding site serves as 11β-hydroxylase, while CYP11B2 with spatially-extended substrate-binding site successively processes additional 18-hydroxylations to produce aldosterone.
AB - Human cytochromes P45011β (CYP11B1) and P450aldo (CYP11B2) are monooxygenases that synthesize cortisol through steroid 11β-hydroxylation and aldosterone through a three-step process comprising 11β-hydroxylation and two 18-hydroxylations, respectively. CYP11B1 also catalyzes 18-monohydroxylation and 11β,18-dihydroxylation. To study the molecular basis of such catalytic divergence of the two enzymes, we examined a CYP11B1 mutant (Mt-CYP11B1) with amino acid replacements on the distal surface by determining the catalytic activities and crystal structure in the metyrapone-bound form at 1.4-Å resolution. Mt-CY11B1 retained both 11β-hydroxylase and 18-hydroxylase activities of the wild type (Wt-CYP11B1) but lacked 11β,18-dihydroxylase activity. Comparisons of the crystal structure of Mt-CYP11B1 to those of Wt-CYP11B1 and CYP11B2 that were already reported show that the mutation reduced the innermost space putatively surrounding the C3 side of substrate 11-deoxycorticosterone (DOC) bound to Wt-CYP11B1, while the corresponding space in CYP11B2 is enlarged markedly and accessible to bulk water through a channel. Molecular dynamics simulations of their DOC-bound forms supported the above findings and revealed that the enlarged space of CYP11B2 had a hydrogen bonding network involving water molecules that position DOC. Thus, upon positioning 11β-hydroxysteroid for 18-hydroxylation in their substrate-binding sites, steric hindrance could occur more strongly in Mt-CYP11B1 than in Wt-CYP11B1 but less in CYP11B2. Our investigation employing Mt-CYP11B1 sheds light on the divergence in structure and function between CYP11B1 and CYP11B2 and suggests that CYP11B1 with spatially-restricted substrate-binding site serves as 11β-hydroxylase, while CYP11B2 with spatially-extended substrate-binding site successively processes additional 18-hydroxylations to produce aldosterone.
KW - Adrenal
KW - Cytochrome P450
KW - Molecular dynamics
KW - Steroid hormone
KW - X-ray crystallography
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U2 - 10.1016/j.crstbi.2021.08.001
DO - 10.1016/j.crstbi.2021.08.001
M3 - Article
AN - SCOPUS:85122694156
VL - 3
SP - 192
EP - 205
JO - Current Research in Structural Biology
JF - Current Research in Structural Biology
SN - 2665-928X
ER -