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J Steroid Biochem Mol Biol. 2014 Oct;144 Pt B:324-33. doi: 10.1016/j.jsbmb.2014.07.005. Epub 2014 Jul 16.

A steroidogenic pathway for sulfonated steroids: the metabolism of pregnenolone sulfate.

Author information

1
Department of Biochemistry, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany.
2
Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine, Justus-Liebig University, 35392 Giessen, Germany.
3
Institute of Pharmacology and Toxicology, Justus-Liebig University of Giessen, 35392 Giessen, Germany.
4
Department of Biochemistry, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany. Electronic address: ritabern@mx.uni-saarland.de.

Abstract

In many tissues sulfonated steroids exceed the concentration of free steroids and recently they were also shown to fulfill important physiological functions. While it was previously demonstrated that cholesterol sulfate (CS) is converted by CYP11A1 to pregnenolone sulfate (PregS), further conversion of PregS has not been studied in detail. To investigate whether a steroidogenic pathway for sulfonated steroids exists similar to the one described for free steroids, we examined the interaction of PregS with CYP17A1 in a reconstituted in-vitro system. Difference spectroscopy revealed a Kd-value of 74.8±4.2μM for the CYP17A1-PregS complex, which is 2.5-fold higher compared to the CYP17A1-pregnenolone (Preg) complex. Mass spectrometry experiments proved for the first time that PregS is hydroxylated by CYP17A1 at position C17, identically to pregnenolone. A higher Km- and a lower kcat-value for CYP17A1 using PregS compared with Preg were observed, indicating a 40% reduced catalytic efficiency when using the sulfonated steroid. Furthermore, we analyzed whether the presence of cytochrome b5 (b5) has an influence on the CYP17A1 dependent conversion of PregS, as was demonstrated for Preg. Interestingly, with 17OH-PregS no scission of the 17,20-carbon-carbon bond occurs, when b5 is added to the reconstituted in-vitro system, while b5 promotes the formation of DHEA from 17OH-Preg. When using human SOAT-HEK293 cells expressing CYP17A1 and CPR, we could confirm that PregS is metabolized to 17OH-PregS, strengthening the potential physiological meaning of a pathway for sulfonated steroids.

KEYWORDS:

CYP17A1; Mass spectrometry; Pregnenolone sulfate; Steroidogenesis; Sulfonated steroids

PMID:
25038322
DOI:
10.1016/j.jsbmb.2014.07.005
[Indexed for MEDLINE]

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