A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate

Neunzig, Jens; Sánchez‐Guijo, Alberto; Mosa, Azzam Abdulsattar; Hartmann, Michaela F.; Geyer, Joachim; Wudy, Stefan A.; Bernhardt, Rita

doi:10.1016/j.jsbmb.2014.07.005

Cited by 38 publications

(14 citation statements)

References 56 publications

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“…The vector pET17b_21b served as a backbone for the construction of the vector p21b_bRED, containing the cDNA for bovine CYP21A2 and its natural redox partner, the bovine cytochrome P450 reductase ( CPR ), as a bicistronic transcription unit. A CPR containing vector was used for PCR amplification of CPR cDNA [ 49 ]. The forward primer contains a BamHI restriction side followed by a ribosomal binding side and the respective coding region.…”

Section: Methodsmentioning

confidence: 99%

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A CYP21A2 based whole-cell system in Escherichia coli for the biotechnological production of premedrol

et al. 2015

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BackgroundSynthetic glucocorticoids like methylprednisolone (medrol) are of high pharmaceutical interest and represent powerful drugs due to their anti-inflammatory and immunosuppressive effects. Since the chemical hydroxylation of carbon atom 21, a crucial step in the synthesis of the medrol precursor premedrol, exhibits a low overall yield because of a poor stereo- and regioselectivity, there is high interest in a more sustainable and efficient biocatalytic process. One promising candidate is the mammalian cytochrome P450 CYP21A2 which is involved in steroid hormone biosynthesis and performs a selective oxyfunctionalization of C21 to provide the precursors of aldosterone, the main mineralocorticoid, and cortisol, the most important glucocorticoid. In this work, we demonstrate the high potential of CYP21A2 for a biotechnological production of premedrol, an important precursor of medrol.ResultsWe successfully developed a CYP21A2-based whole-cell system in Escherichia coli by coexpressing the cDNAs of bovine CYP21A2 and its redox partner, the NADPH-dependent cytochrome P450 reductase (CPR), via a bicistronic vector. The synthetic substrate medrane was selectively 21-hydroxylated to premedrol with a max. yield of 90 mg L−1 d−1. To further improve the biocatalytic activity of the system by a more effective electron supply, we exchanged the CPR with constructs containing five alternative redox systems. A comparison of the constructs revealed that the redox system with the highest endpoint yield converted 70 % of the substrate within the first 2 h showing a doubled initial reaction rate compared with the other constructs. Using the best system we could increase the overall yield of premedrol to a maximum of 320 mg L−1 d−1 in shaking flasks. Optimization of the biotransformation in a bioreactor could further improve the premedrol gain to a maximum of 0.65 g L−1 d−1.ConclusionsWe successfully established a CYP21-based whole-cell system for the biotechnological production of premedrol, a pharmaceutically relevant glucocorticoid, in E. coli and could improve the system by optimizing the redox system concerning reaction velocity and endpoint yield. This is the first step for a sustainable replacement of a complicated chemical low-yield hydroxylation by a biocatalytic cytochrome P450-based whole-cell system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0333-2) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

“…Bovine CPR was synthesized in E. coli and purified via Immobilized Metal Ion Affinity Chromatography (IMAC) as described elsewhere [ 49 ].…”

Section: Methodsmentioning

confidence: 99%

A CYP21A2 based whole-cell system in Escherichia coli for the biotechnological production of premedrol

et al. 2015

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“…35 The side chain cleavage of cholesterol by CYP11A1 is performed 36 through three consecutive steps: two hydroxylations at C22 and 37 C20, respectively, followed by the cleavage of the C20-C22 bond 38 [2,10,13]. The resulting product, pregnenolone, is the precursor of 39 all classes of steroid hormones: mineralocorticoids, glucocorti- 40 coids and sex hormones [14,15]. Therefore, CYP11A1 might 41 represent a vital target for the regulation of steroidogenesis at 42 the cellular level.…”

Section: Introductionmentioning

confidence: 99%

2β- and 16β-hydroxylase activity of CYP11A1 and direct stimulatory effect of estrogens on pregnenolone formation

Mosa

Neunzig

Gerber

et al. 2015

The Journal of Steroid Biochemistry and Molecular Biology

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“…The concept that sulfation does not prevent downstream conversion of steroids, but modulates it, is based on the side-chain-cleaving activity of cytochrome P450 CYP11A1 toward cholesterol sulfate (Tuckey 1990). This observation was then extended to CYP17A1 that bound and metabolized pregnenolone sulfate (Neunzig et al 2014). It is STS that can then convert sulfated steroids to biologically active steroids (Sanchez-Guijo et al 2016).…”

Section: Journal Of Molecular Endocrinologymentioning

confidence: 98%

SULFATION PATHWAYS: Insights into steroid sulfation and desulfation pathways

Foster

Mueller

2018

Journal of Molecular Endocrinology

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Sulfation and desulfation pathways represent highly dynamic ways of shuttling, repressing and re-activating steroid hormones, thus controlling their immense biological potency at the very heart of endocrinology. This theme currently experiences growing research interest from various sides, including, but not limited to, novel insights about phospho-adenosine-5'-phosphosulfate synthase and sulfotransferase function and regulation, novel analytics for steroid conjugate detection and quantification. Within this review, we will also define how sulfation pathways are ripe for drug development strategies, which have translational potential to treat a number of conditions, including chronic inflammatory diseases and steroid-dependent cancers.

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A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate

Cited by 38 publications

References 56 publications

A CYP21A2 based whole-cell system in Escherichia coli for the biotechnological production of premedrol

A CYP21A2 based whole-cell system in Escherichia coli for the biotechnological production of premedrol

2β- and 16β-hydroxylase activity of CYP11A1 and direct stimulatory effect of estrogens on pregnenolone formation

SULFATION PATHWAYS: Insights into steroid sulfation and desulfation pathways

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