TY - JOUR
T1 - P450 CYP17A1 Variant with a Disordered Proton Shuttle Assembly Retains Peroxo-Mediated Lyase Efficiency
AU - Liu, Yilin
AU - Denisov, Ilia G.
AU - Grinkova, Yelena V.
AU - Sligar, Stephen G.
AU - Kincaid, James R.
N1 - Funding Information:
We acknowledge grant support from the National Institutes of Health, MIRA R35GM118145 (S.G.S.), R01 GM110428 (J.R.K.) and R01 GM125303 (J.R.K.). We appreciate the help provided by Dr. Jay A. LaVerne, Notre Dame Radiation Laboratory (Notre Dame University, IN), a facility of the US Department of Energy, Office of Basic Energy Science.
Funding Information:
We acknowledge grant support from the National Institutes of Health, MIRA R35GM118145 (S.G.S.), R01 GM110428 (J.R.K.) and R01 GM125303 (J.R.K.). We appreciate the help provided by Dr. Jay A. LaVerne, Notre Dame Radiation Laboratory (Notre Dame University, IN), a facility of the US Department of Energy, Office of Basic Energy Science.
Publisher Copyright:
© 2020 Wiley-VCH GmbH
PY - 2020/12/15
Y1 - 2020/12/15
N2 - Human cytochrome P450 CYP17A1 first catalyzes hydroxylation at the C17 position of either pregnenolone (PREG) or progesterone (PROG), and a subsequent C17−C20 bond scission to produce dehydroepiandrosterone (DHEA) or androstenedione (AD). In the T306A mutant, replacement of the Threonine 306 alcohol functionality, essential for efficient proton delivery in the hydroxylase reaction, has only a small effect on the lyase activity. In this work, resonance Raman spectroscopy is employed to provide crucial structural insight, confirming that this mutant, with its disordered proton shuttle, fails to generate essential hydroxylase pathway intermediates, accounting for the loss in hydroxylase efficiency. Significantly, a corresponding spectroscopic study with the susceptible lyase substrate, 17-OH PREG, not only reveals an initially trapped peroxo-iron intermediate experiencing an H-bond interaction of the 17-OH group with the proximal oxygen of the Fe-Op-Ot fragment, facilitating peroxo- attack on the C20 carbon, but also unequivocally shows the presence of the subsequent hemiketal intermediate of the lyase reaction.
AB - Human cytochrome P450 CYP17A1 first catalyzes hydroxylation at the C17 position of either pregnenolone (PREG) or progesterone (PROG), and a subsequent C17−C20 bond scission to produce dehydroepiandrosterone (DHEA) or androstenedione (AD). In the T306A mutant, replacement of the Threonine 306 alcohol functionality, essential for efficient proton delivery in the hydroxylase reaction, has only a small effect on the lyase activity. In this work, resonance Raman spectroscopy is employed to provide crucial structural insight, confirming that this mutant, with its disordered proton shuttle, fails to generate essential hydroxylase pathway intermediates, accounting for the loss in hydroxylase efficiency. Significantly, a corresponding spectroscopic study with the susceptible lyase substrate, 17-OH PREG, not only reveals an initially trapped peroxo-iron intermediate experiencing an H-bond interaction of the 17-OH group with the proximal oxygen of the Fe-Op-Ot fragment, facilitating peroxo- attack on the C20 carbon, but also unequivocally shows the presence of the subsequent hemiketal intermediate of the lyase reaction.
KW - P450 CYP17A1
KW - T306A mutant
KW - cryoradiolysis
KW - proton shuttle
KW - resonance Raman spectroscopy
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U2 - 10.1002/chem.202003181
DO - 10.1002/chem.202003181
M3 - Article
C2 - 32681807
AN - SCOPUS:85096687251
SN - 0947-6539
VL - 26
SP - 16846
EP - 16852
JO - Chemistry - A European Journal
JF - Chemistry - A European Journal
IS - 70
ER -