Human P450 CYP17A1: Control of Substrate Preference by Asparagine 202

Michael C. Gregory, Piotr J. Mak, Yogan Khatri, James R. Kincaid, Stephen G. Sligar

Research output: Contribution to journalArticle

Abstract

CYP17A1 is a key steroidogenic enzyme known to conduct several distinct chemical transformations on multiple substrates. In its hydroxylase activity, this enzyme adds a hydroxyl group at the 17α position of both pregnenolone and progesterone at approximately equal rates. However, the subsequent 17,20 carbon-carbon scission reaction displays variable substrate specificity in the numerous CYP17A1 isozymes operating in vertebrates, manifesting as different Kd and kcat values when presented with 17α-hydroxypregnenlone (OHPREG) versus 17α-hydroxyprogesterone (OHPROG). Here we show that the identity of the residue at position 202 in human CYP17A1, thought to form a hydrogen bond with the A-ring alcohol substituent on the pregnene- nucleus, is a key driver of this enzyme's native preference for OHPREG. Replacement of asparagine 202 with serine completely reverses the preference of CYP17A1, more than doubling the rate of turnover of the OHPROG to androstenedione reaction and substantially decreasing the rate of formation of dehydroepiandrosterone from OHPREG. In a series of resonance Raman experiments, it was observed that, in contrast with the case for the wild-type protein, in the mutant the 17α alcohol of OHPROG tends to form a H-bond with the proximal rather than terminal oxygen of the oxy-ferrous complex. When OHPREG was a substrate, the mutant enzyme was found to have a H-bonding interaction with the proximal oxygen that is substantially weaker than that of the wild type. These results demonstrate that a single-point mutation in the active site pocket of CYP17A1, even when far from the heme, has profound effects on steroidogenic selectivity in androgen biosynthesis.

Original languageEnglish (US)
Pages (from-to)764-771
Number of pages8
JournalBiochemistry
Volume57
Issue number5
DOIs
StatePublished - Feb 6 2018

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17-alpha-Hydroxyprogesterone
Asparagine
Substrates
Enzymes
Pregnenes
Carbon
Alcohols
Oxygen
Pregnenolone
Dehydroepiandrosterone
Androstenedione
Biosynthesis
Enzyme activity
Mixed Function Oxygenases
Heme
Hydroxyl Radical
Serine
Androgens
Isoenzymes
Progesterone

ASJC Scopus subject areas

  • Biochemistry

Cite this

Human P450 CYP17A1 : Control of Substrate Preference by Asparagine 202. / Gregory, Michael C.; Mak, Piotr J.; Khatri, Yogan; Kincaid, James R.; Sligar, Stephen G.

In: Biochemistry, Vol. 57, No. 5, 06.02.2018, p. 764-771.

Research output: Contribution to journalArticle

Gregory, Michael C. ; Mak, Piotr J. ; Khatri, Yogan ; Kincaid, James R. ; Sligar, Stephen G. / Human P450 CYP17A1 : Control of Substrate Preference by Asparagine 202. In: Biochemistry. 2018 ; Vol. 57, No. 5. pp. 764-771.
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