Properties of purified CYP2R1 in a reconstituted membrane environment and its 25-hydroxylation of 20-hydroxyvitamin D3

Chloe Y.S. Cheng, Tae Kang Kim, Saowanee Jeayeng, Andrzej T. Slominski, Robert C. Tuckey

Research output: Contribution to journalArticle

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Abstract

Recent studies indicate that CYP2R1 is the major 25-hydroxylase catalyzing the first step in vitamin D activation. Since the catalytic properties of CYP2R1 have been poorly studied to date and it is a membrane protein, we examined the purified enzyme in a membrane environment. CYP2R1 was expressed in E. coli and purified by nickel affinity- and hydrophobic interaction-chromatography and assayed in a reconstituted membrane system comprising phospholipid vesicles plus purified human NADPH-P450 oxidoreductase. CYP2R1 converted vitamin D3 in the vesicle membrane to 25-hydroxyvitamin D3 [25(OH)D3] with good adherence to Michaelis-Menten kinetics. The kinetic parameters for 25-hydroxylation of vitamin D3 by the two major vitamin D 25-hydroxylases, CYP2R1 and CYP27A1, were examined in vesicles under identical conditions. CYP2R1 displayed a slightly lower kcat than CYP27A1 but a much lower Km for vitamin D3, and thus an overall 17-fold higher catalytic efficiency (kcat/Km), consistent with CYP2R1 being the major vitamin D 25-hydroxylase. 20-Hydroxyvitamin D3 [20(OH)D3], the main product of vitamin D3 activation by an alternative pathway catalyzed by CYP11A1, was metabolized by CYP2R1 to 20,25-dihydroxyvitamin D3 [20,25(OH)2D3], with catalytic efficiency similar to that for the 25-hydroxylation of vitamin D3. 20,25(OH)2D3 retained full, or somewhat enhanced activity compared to the parent 20(OH)D3 for the inhibition of the proliferation of melanocytes and dermal fibroblasts, with a potency comparable to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The 20,25(OH)2D3 was also able to act as an inverse agonist on retinoic acid-related orphan receptor α, like its parent 20(OH)D3. Thus, the major findings of this study are that CYP2R1 can metabolize substrates in a membrane environment, the enzyme displays higher catalytic efficiency than CYP27A1 for the 25-hydroxylation of vitamin D, it efficiently hydroxylates 20(OH)D3 at C25 and this product retains the biological activity of the parent compound.

Original languageEnglish
Pages (from-to)59-69
Number of pages11
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume177
DOIs
Publication statusPublished - 1 Mar 2018

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Hydroxylation
Cholecalciferol
Vitamin D
Membranes
Mixed Function Oxygenases
Chemical activation
Cholesterol Side-Chain Cleavage Enzyme
Calcifediol
Calcitriol
Melanocytes
Enzymes
Fibroblasts
Chromatography
Tretinoin
Bioactivity
Nickel
NADP
Hydrophobic and Hydrophilic Interactions
Kinetic parameters
Escherichia coli

Cite this

@article{efb4e3799b62475ca610e7bbcdc06a40,
title = "Properties of purified CYP2R1 in a reconstituted membrane environment and its 25-hydroxylation of 20-hydroxyvitamin D3",
abstract = "Recent studies indicate that CYP2R1 is the major 25-hydroxylase catalyzing the first step in vitamin D activation. Since the catalytic properties of CYP2R1 have been poorly studied to date and it is a membrane protein, we examined the purified enzyme in a membrane environment. CYP2R1 was expressed in E. coli and purified by nickel affinity- and hydrophobic interaction-chromatography and assayed in a reconstituted membrane system comprising phospholipid vesicles plus purified human NADPH-P450 oxidoreductase. CYP2R1 converted vitamin D3 in the vesicle membrane to 25-hydroxyvitamin D3 [25(OH)D3] with good adherence to Michaelis-Menten kinetics. The kinetic parameters for 25-hydroxylation of vitamin D3 by the two major vitamin D 25-hydroxylases, CYP2R1 and CYP27A1, were examined in vesicles under identical conditions. CYP2R1 displayed a slightly lower kcat than CYP27A1 but a much lower Km for vitamin D3, and thus an overall 17-fold higher catalytic efficiency (kcat/Km), consistent with CYP2R1 being the major vitamin D 25-hydroxylase. 20-Hydroxyvitamin D3 [20(OH)D3], the main product of vitamin D3 activation by an alternative pathway catalyzed by CYP11A1, was metabolized by CYP2R1 to 20,25-dihydroxyvitamin D3 [20,25(OH)2D3], with catalytic efficiency similar to that for the 25-hydroxylation of vitamin D3. 20,25(OH)2D3 retained full, or somewhat enhanced activity compared to the parent 20(OH)D3 for the inhibition of the proliferation of melanocytes and dermal fibroblasts, with a potency comparable to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The 20,25(OH)2D3 was also able to act as an inverse agonist on retinoic acid-related orphan receptor α, like its parent 20(OH)D3. Thus, the major findings of this study are that CYP2R1 can metabolize substrates in a membrane environment, the enzyme displays higher catalytic efficiency than CYP27A1 for the 25-hydroxylation of vitamin D, it efficiently hydroxylates 20(OH)D3 at C25 and this product retains the biological activity of the parent compound.",
keywords = "25-hydroxylase, CYP2R1, Phospholipid vesicles, Vitamin D3",
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Properties of purified CYP2R1 in a reconstituted membrane environment and its 25-hydroxylation of 20-hydroxyvitamin D3. / Cheng, Chloe Y.S.; Kim, Tae Kang; Jeayeng, Saowanee; Slominski, Andrzej T.; Tuckey, Robert C.

In: Journal of Steroid Biochemistry and Molecular Biology, Vol. 177, 01.03.2018, p. 59-69.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Properties of purified CYP2R1 in a reconstituted membrane environment and its 25-hydroxylation of 20-hydroxyvitamin D3

AU - Cheng, Chloe Y.S.

AU - Kim, Tae Kang

AU - Jeayeng, Saowanee

AU - Slominski, Andrzej T.

AU - Tuckey, Robert C.

PY - 2018/3/1

Y1 - 2018/3/1

N2 - Recent studies indicate that CYP2R1 is the major 25-hydroxylase catalyzing the first step in vitamin D activation. Since the catalytic properties of CYP2R1 have been poorly studied to date and it is a membrane protein, we examined the purified enzyme in a membrane environment. CYP2R1 was expressed in E. coli and purified by nickel affinity- and hydrophobic interaction-chromatography and assayed in a reconstituted membrane system comprising phospholipid vesicles plus purified human NADPH-P450 oxidoreductase. CYP2R1 converted vitamin D3 in the vesicle membrane to 25-hydroxyvitamin D3 [25(OH)D3] with good adherence to Michaelis-Menten kinetics. The kinetic parameters for 25-hydroxylation of vitamin D3 by the two major vitamin D 25-hydroxylases, CYP2R1 and CYP27A1, were examined in vesicles under identical conditions. CYP2R1 displayed a slightly lower kcat than CYP27A1 but a much lower Km for vitamin D3, and thus an overall 17-fold higher catalytic efficiency (kcat/Km), consistent with CYP2R1 being the major vitamin D 25-hydroxylase. 20-Hydroxyvitamin D3 [20(OH)D3], the main product of vitamin D3 activation by an alternative pathway catalyzed by CYP11A1, was metabolized by CYP2R1 to 20,25-dihydroxyvitamin D3 [20,25(OH)2D3], with catalytic efficiency similar to that for the 25-hydroxylation of vitamin D3. 20,25(OH)2D3 retained full, or somewhat enhanced activity compared to the parent 20(OH)D3 for the inhibition of the proliferation of melanocytes and dermal fibroblasts, with a potency comparable to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The 20,25(OH)2D3 was also able to act as an inverse agonist on retinoic acid-related orphan receptor α, like its parent 20(OH)D3. Thus, the major findings of this study are that CYP2R1 can metabolize substrates in a membrane environment, the enzyme displays higher catalytic efficiency than CYP27A1 for the 25-hydroxylation of vitamin D, it efficiently hydroxylates 20(OH)D3 at C25 and this product retains the biological activity of the parent compound.

AB - Recent studies indicate that CYP2R1 is the major 25-hydroxylase catalyzing the first step in vitamin D activation. Since the catalytic properties of CYP2R1 have been poorly studied to date and it is a membrane protein, we examined the purified enzyme in a membrane environment. CYP2R1 was expressed in E. coli and purified by nickel affinity- and hydrophobic interaction-chromatography and assayed in a reconstituted membrane system comprising phospholipid vesicles plus purified human NADPH-P450 oxidoreductase. CYP2R1 converted vitamin D3 in the vesicle membrane to 25-hydroxyvitamin D3 [25(OH)D3] with good adherence to Michaelis-Menten kinetics. The kinetic parameters for 25-hydroxylation of vitamin D3 by the two major vitamin D 25-hydroxylases, CYP2R1 and CYP27A1, were examined in vesicles under identical conditions. CYP2R1 displayed a slightly lower kcat than CYP27A1 but a much lower Km for vitamin D3, and thus an overall 17-fold higher catalytic efficiency (kcat/Km), consistent with CYP2R1 being the major vitamin D 25-hydroxylase. 20-Hydroxyvitamin D3 [20(OH)D3], the main product of vitamin D3 activation by an alternative pathway catalyzed by CYP11A1, was metabolized by CYP2R1 to 20,25-dihydroxyvitamin D3 [20,25(OH)2D3], with catalytic efficiency similar to that for the 25-hydroxylation of vitamin D3. 20,25(OH)2D3 retained full, or somewhat enhanced activity compared to the parent 20(OH)D3 for the inhibition of the proliferation of melanocytes and dermal fibroblasts, with a potency comparable to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The 20,25(OH)2D3 was also able to act as an inverse agonist on retinoic acid-related orphan receptor α, like its parent 20(OH)D3. Thus, the major findings of this study are that CYP2R1 can metabolize substrates in a membrane environment, the enzyme displays higher catalytic efficiency than CYP27A1 for the 25-hydroxylation of vitamin D, it efficiently hydroxylates 20(OH)D3 at C25 and this product retains the biological activity of the parent compound.

KW - 25-hydroxylase

KW - CYP2R1

KW - Phospholipid vesicles

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U2 - 10.1016/j.jsbmb.2017.07.011

DO - 10.1016/j.jsbmb.2017.07.011

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EP - 69

JO - Journal of Steroid Biochemistry & Molecular Biology

JF - Journal of Steroid Biochemistry & Molecular Biology

SN - 0960-0760

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