Structural characterization of human cholesterol 7α-hydroxylase

Tempel, W.; Grabovec, Irina; Mackenzie, F.; Dichenko, Yaroslav V.; Usanov, Sergey A.; Gilep, Andrei A.; Park, Hee-Won; Strushkevich, Natallia

doi:10.1194/jlr.m050765

Cited by 29 publications

(29 citation statements)

References 52 publications

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“…[2b,24] P450 difference spectra were recorded as described, [25] except two double, not single, cuvettes were used to compensate for a residual hydroperoxide absorption in the visible region. The concentrations of P450 and cholesterol hydroperoxide were 5 μM and 10 μM, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…[1] These enzymes include the 7-, 24- and 27-hydroxylases (CYP7A1, CYP46A1 and CYP27A1, respectively) as well as the cholesterol side-chain cleavage enzyme CYP11A1. [1,2] CYP7A1 and CYP46A1 are microsomal enzymes, whereas CYP27A1 and CYP11A1 are mitochondrial monooxygenases. CYP7A1 is expressed only in the liver, CYP46A1 is mainly found in the neurons, CYP27A1 is ubiquitous, and CYP11A1 is the main cholesterol hydroxylase in steroidogenic tissues.…”

mentioning

confidence: 99%

“…The stereospecific 20ξ-hydroperoxide-glycol reactions can be envisaged by substituting 22R-hydroxycholesterol with 20α-hydroperoxide 2 or 20β-hydroperoxide 3 in the active site of the previously determined 22R-hydroxycholesterol-CYP11A1 co-complex [2b] (Figure 2). The model shows the hydroperoxide oxygen of 2 at a distances of 2.7 Å from the Fe(III) haem iron allowing formation of the transient haem-Fe(IV)=O +• species (Cpd I) that subsequently can oxidize the adjacent 22R-position of 2 (4 Å from the haem iron) to yield the 20α,22R-glycol 7 .…”

mentioning

confidence: 99%

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Cholesterol Hydroperoxides as Substrates for Cholesterol‐Metabolizing Cytochrome P450 Enzymes and Alternative Sources of 25‐Hydroxycholesterol and other Oxysterols

Lier

Mast²,

Pikuleva

2015

Angew Chem Int Ed

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The interaction of primary autoxidation products of cholesterol, 25- and 20ξ-hydroperoxides, with the four principal cholesterol-metabolizing cytochrome P450 enzymes is reported. Addition of cholesterol 25-hydroperoxide to CYP27A1 and CYP11A1 induced well-defined spectral changes while generating 25-hydroxycholesterol as major product along with small amounts of triols. The 20ξ-hydroperoxides induced spectral shifts in CYP27A1 and CYP11A1, yet glycol metabolites were detected only with CYP11A1. CYP7A1 and CYP46A1 failed to give metabolites with any of the hydroperoxides. A P450 hydroperoxide-shunt reaction is proposed, where the hydroperoxides serve both as donor for reduced oxygen and as substrate. For the first time, CYP27A1 is shown to mediate the reduction of cholesterol 25-hydroperoxide to 25-hydroxycholesterol, a role of potential significance for cholesterol-rich tissues with high oxidative stress. CYP27A1 may participate in these tissues in removal of harmful autoxidation products, while providing a complementary source for 25-hydroxycholesterol, a modulator of immune cell function and mediator of viral cell entry.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Cholesterol Hydroperoxides as Substrates for Cholesterol‐Metabolizing Cytochrome P450 Enzymes and Alternative Sources of 25‐Hydroxycholesterol and other Oxysterols

Lier

Mast²,

Pikuleva

2015

Angew Chem Int Ed

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“…Comparative modeling in Rosetta predicts protein structure based on solved structures of homologs, from which it generates candidate structures. CYP7A1, which is 41% sequence identical to CYP7B1 with 95% coverage, has been crystallized and served as a starting point for comparative modeling 18 . To account for induced fit in the active site, only .…”

Section: Cc-by-nc-ndmentioning

confidence: 99%

“…This resulted in 0. BLAST confidence, and bound to chemically similar substrates, were PDB IDs 3sn5 and 3v8d 18 .…”

Section: Protein Expression In Mammalianmentioning

confidence: 99%

Mammalian Cells Engineered to Produce Novel Steroids

Spady

Wyche

Rollins

et al. 2018

Preprint

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Steroids can be difficult to modify via traditional organic synthesis methods, but many enzymes regio-and stereo-selectively process a wide variety of steroid substrates. We tested whether steroid-modifying enzymes could make novel steroids from non-native substrates. Numerous genes encoding steroid-modifying enzymes, including some bacterial enzymes, were expressed in mammalian cells by transient transfection and found to be active. We made three unusual steroids by expression in HEK293 cells of the 7α-hydroxylase CYP7B1, which was selected because of high native product yield. These cells made 7α,17α-dihydroxypregnenolone and 7β,17α-dihydroxypregnenolone from 17α-hydroxypregnenolone, and produced 11α,16α-dihydroxyprogesterone from 16α-hydroxyprogesterone. The latter two products resulted from previously unobserved CYP7B1 hydroxylation sites. A Rosetta docking model of CYP7B1 suggested that these substrates' D-ring hydroxylations may prevent them from binding in the same way as the native substrate, bringing different carbons near the active ferryl oxygen. This new approach could use other enzymes and substrates to produce many novel steroids for drug candidate testing.

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Cholesterol Hydroperoxides as Substrates for Cholesterol‐Metabolizing Cytochrome P450 Enzymes and Alternative Sources of 25‐Hydroxycholesterol and other Oxysterols

Lier

Mast²,

Pikuleva

2015

Angewandte Chemie

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The interaction of the primary autoxidation products of cholesterol, namely 25-and 20x-hydroperoxides,w ith the four principal cholesterol-metabolizing cytochrome P450 enzymes is reported. Addition of cholesterol 25-hydroperoxide to the enzymes CYP27A1 and CYP11A1 induced well-defined spectral changes while generating 25-hydroxycholesterol as the major product. The 20x-hydroperoxides induced spectral shifts in CYP27A1 and CYP11A1 but glycol metabolites were detected only with CYP11A1. CYP7A1 and CYP46A1 failed to give metabolites with any of the hydroperoxides.AP450 hydroperoxide-shunt reaction is proposed, where the hydroperoxides serve as both donor for reduced oxygen and substrate.C YP27A1 was shown to mediate the reduction of cholesterol 25-hydroperoxide to 25-hydroxycholesterol, ar ole of potential significance for cholesterol-richt issues with high oxidative stress.C YP27A1 may participate in the removal of harmful autoxidation products in these tissues,while providing ac omplementary source of 25-hydroxycholesterol, am odulator of immune cell function and mediator of viral cell entry.

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Structural characterization of human cholesterol 7α-hydroxylase

Cited by 29 publications

References 52 publications

Cholesterol Hydroperoxides as Substrates for Cholesterol‐Metabolizing Cytochrome P450 Enzymes and Alternative Sources of 25‐Hydroxycholesterol and other Oxysterols

Cholesterol Hydroperoxides as Substrates for Cholesterol‐Metabolizing Cytochrome P450 Enzymes and Alternative Sources of 25‐Hydroxycholesterol and other Oxysterols

Mammalian Cells Engineered to Produce Novel Steroids

Cholesterol Hydroperoxides as Substrates for Cholesterol‐Metabolizing Cytochrome P450 Enzymes and Alternative Sources of 25‐Hydroxycholesterol and other Oxysterols

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