Catalytic properties of 25-hydroxyvitamin D3 3-epimerase in rat and human liver microsomes

Robert C. Tuckey, Edith K.Y. Tang, Stephanie R. Maresse, Derek S. Delaney

Research output: Contribution to journalArticle

Abstract

25-Hydroxyvitamin D3 3-epimerase catalyzes the 3β → 3α epimerization of 25-hydroxyvitamin D3 (25(OH)D3) producing 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3). 3-Epi-25(OH)D3 is one of the most abundant forms of vitamin D present in the serum. It can be converted to 3-epi-1α,25-dihydroxyvitamin D3 by CYP27B1 which generally displays lower biological activity than 1α,25-dihydroxyvitamin D3 (1,25(OH) 2 D3). The 25(OH)D3 3-epimerase has been poorly characterized to date and the gene encoding it has not been identified. The 3-epimerase has been reported to be present in the microsomal fraction of cells, including liver cells, and to use NADPH as cofactor. It can also act on 1,25(OH) 2 D3 and 24,25(OH) 2 D3 forming the 3α-epimers. In this study we have characterized the activity of the 25(OH)D3 3-epimerase in rat and human liver microsomes, using 25(OH)D3 as substrate and HPLC to analyze product formation. For both rat and human liver microsomes the preferred cofactor was NADH, with the rat enzyme displaying a 6-fold greater catalytic efficiency (V max /K m ) for NADH over that for NADPH. No activity was observed with oxidized cofactor, either NAD + or NADP + . This was unexpected since the initial step in the epimerization, predicted to be the oxidation of the 3β-OH to a ketone, would require oxidized cofactor. The rat 3-epimerase in microsomes gave a K m for 25(OH)D3 of 14 μM. The reverse reaction, conversion of 3-epi-25(OH)D3 to 25(OH)D3, was catalyzed by both rat and human liver microsomes but at lower rates than the forward reaction. In conclusion, both rat and human 25-hydroxyvitamin D3 3-epimerase catalyze the reversible interconversion of 25(OH)D3 and 3-epi-25(OH)D3, and use NADH as the preferred cofactor. The lack of requirement for exogenous NAD + suggests that the enzyme has a tightly bound NAD + in its active site that is released only upon its reduction.

Original languageEnglish
Pages (from-to)16-21
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume666
DOIs
Publication statusPublished - 15 May 2019

Fingerprint

Racemases and Epimerases
Calcifediol
Liver Microsomes
Liver
NAD
Rats
NADP
Calcitriol
25-Hydroxyvitamin D3 1-alpha-Hydroxylase
Conversion Disorder
Gene encoding
Enzymes
Microsomes
Bioactivity
Ketones
Vitamin D
Catalytic Domain
High Pressure Liquid Chromatography
Oxidation
Substrates

Cite this

@article{3379aaf8e23547859f6c2341cdcd931d,
title = "Catalytic properties of 25-hydroxyvitamin D3 3-epimerase in rat and human liver microsomes",
abstract = "25-Hydroxyvitamin D3 3-epimerase catalyzes the 3β → 3α epimerization of 25-hydroxyvitamin D3 (25(OH)D3) producing 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3). 3-Epi-25(OH)D3 is one of the most abundant forms of vitamin D present in the serum. It can be converted to 3-epi-1α,25-dihydroxyvitamin D3 by CYP27B1 which generally displays lower biological activity than 1α,25-dihydroxyvitamin D3 (1,25(OH) 2 D3). The 25(OH)D3 3-epimerase has been poorly characterized to date and the gene encoding it has not been identified. The 3-epimerase has been reported to be present in the microsomal fraction of cells, including liver cells, and to use NADPH as cofactor. It can also act on 1,25(OH) 2 D3 and 24,25(OH) 2 D3 forming the 3α-epimers. In this study we have characterized the activity of the 25(OH)D3 3-epimerase in rat and human liver microsomes, using 25(OH)D3 as substrate and HPLC to analyze product formation. For both rat and human liver microsomes the preferred cofactor was NADH, with the rat enzyme displaying a 6-fold greater catalytic efficiency (V max /K m ) for NADH over that for NADPH. No activity was observed with oxidized cofactor, either NAD + or NADP + . This was unexpected since the initial step in the epimerization, predicted to be the oxidation of the 3β-OH to a ketone, would require oxidized cofactor. The rat 3-epimerase in microsomes gave a K m for 25(OH)D3 of 14 μM. The reverse reaction, conversion of 3-epi-25(OH)D3 to 25(OH)D3, was catalyzed by both rat and human liver microsomes but at lower rates than the forward reaction. In conclusion, both rat and human 25-hydroxyvitamin D3 3-epimerase catalyze the reversible interconversion of 25(OH)D3 and 3-epi-25(OH)D3, and use NADH as the preferred cofactor. The lack of requirement for exogenous NAD + suggests that the enzyme has a tightly bound NAD + in its active site that is released only upon its reduction.",
keywords = "25-Hydroxyvitamin D3, 3-Epi-25-hydroxyvitamin D3, Epimerase, Hydroxysteroid dehydrogenase, Microsomes",
author = "Tuckey, {Robert C.} and Tang, {Edith K.Y.} and Maresse, {Stephanie R.} and Delaney, {Derek S.}",
year = "2019",
month = "5",
day = "15",
doi = "10.1016/j.abb.2019.03.010",
language = "English",
volume = "666",
pages = "16--21",
journal = "Archives of Biochem and Biophysics",
issn = "0003-9861",
publisher = "Academic Press",

}

Catalytic properties of 25-hydroxyvitamin D3 3-epimerase in rat and human liver microsomes. / Tuckey, Robert C.; Tang, Edith K.Y.; Maresse, Stephanie R.; Delaney, Derek S.

In: Archives of Biochemistry and Biophysics, Vol. 666, 15.05.2019, p. 16-21.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Catalytic properties of 25-hydroxyvitamin D3 3-epimerase in rat and human liver microsomes

AU - Tuckey, Robert C.

AU - Tang, Edith K.Y.

AU - Maresse, Stephanie R.

AU - Delaney, Derek S.

PY - 2019/5/15

Y1 - 2019/5/15

N2 - 25-Hydroxyvitamin D3 3-epimerase catalyzes the 3β → 3α epimerization of 25-hydroxyvitamin D3 (25(OH)D3) producing 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3). 3-Epi-25(OH)D3 is one of the most abundant forms of vitamin D present in the serum. It can be converted to 3-epi-1α,25-dihydroxyvitamin D3 by CYP27B1 which generally displays lower biological activity than 1α,25-dihydroxyvitamin D3 (1,25(OH) 2 D3). The 25(OH)D3 3-epimerase has been poorly characterized to date and the gene encoding it has not been identified. The 3-epimerase has been reported to be present in the microsomal fraction of cells, including liver cells, and to use NADPH as cofactor. It can also act on 1,25(OH) 2 D3 and 24,25(OH) 2 D3 forming the 3α-epimers. In this study we have characterized the activity of the 25(OH)D3 3-epimerase in rat and human liver microsomes, using 25(OH)D3 as substrate and HPLC to analyze product formation. For both rat and human liver microsomes the preferred cofactor was NADH, with the rat enzyme displaying a 6-fold greater catalytic efficiency (V max /K m ) for NADH over that for NADPH. No activity was observed with oxidized cofactor, either NAD + or NADP + . This was unexpected since the initial step in the epimerization, predicted to be the oxidation of the 3β-OH to a ketone, would require oxidized cofactor. The rat 3-epimerase in microsomes gave a K m for 25(OH)D3 of 14 μM. The reverse reaction, conversion of 3-epi-25(OH)D3 to 25(OH)D3, was catalyzed by both rat and human liver microsomes but at lower rates than the forward reaction. In conclusion, both rat and human 25-hydroxyvitamin D3 3-epimerase catalyze the reversible interconversion of 25(OH)D3 and 3-epi-25(OH)D3, and use NADH as the preferred cofactor. The lack of requirement for exogenous NAD + suggests that the enzyme has a tightly bound NAD + in its active site that is released only upon its reduction.

AB - 25-Hydroxyvitamin D3 3-epimerase catalyzes the 3β → 3α epimerization of 25-hydroxyvitamin D3 (25(OH)D3) producing 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3). 3-Epi-25(OH)D3 is one of the most abundant forms of vitamin D present in the serum. It can be converted to 3-epi-1α,25-dihydroxyvitamin D3 by CYP27B1 which generally displays lower biological activity than 1α,25-dihydroxyvitamin D3 (1,25(OH) 2 D3). The 25(OH)D3 3-epimerase has been poorly characterized to date and the gene encoding it has not been identified. The 3-epimerase has been reported to be present in the microsomal fraction of cells, including liver cells, and to use NADPH as cofactor. It can also act on 1,25(OH) 2 D3 and 24,25(OH) 2 D3 forming the 3α-epimers. In this study we have characterized the activity of the 25(OH)D3 3-epimerase in rat and human liver microsomes, using 25(OH)D3 as substrate and HPLC to analyze product formation. For both rat and human liver microsomes the preferred cofactor was NADH, with the rat enzyme displaying a 6-fold greater catalytic efficiency (V max /K m ) for NADH over that for NADPH. No activity was observed with oxidized cofactor, either NAD + or NADP + . This was unexpected since the initial step in the epimerization, predicted to be the oxidation of the 3β-OH to a ketone, would require oxidized cofactor. The rat 3-epimerase in microsomes gave a K m for 25(OH)D3 of 14 μM. The reverse reaction, conversion of 3-epi-25(OH)D3 to 25(OH)D3, was catalyzed by both rat and human liver microsomes but at lower rates than the forward reaction. In conclusion, both rat and human 25-hydroxyvitamin D3 3-epimerase catalyze the reversible interconversion of 25(OH)D3 and 3-epi-25(OH)D3, and use NADH as the preferred cofactor. The lack of requirement for exogenous NAD + suggests that the enzyme has a tightly bound NAD + in its active site that is released only upon its reduction.

KW - 25-Hydroxyvitamin D3

KW - 3-Epi-25-hydroxyvitamin D3

KW - Epimerase

KW - Hydroxysteroid dehydrogenase

KW - Microsomes

UR - http://www.scopus.com/inward/record.url?scp=85063537720&partnerID=8YFLogxK

U2 - 10.1016/j.abb.2019.03.010

DO - 10.1016/j.abb.2019.03.010

M3 - Article

VL - 666

SP - 16

EP - 21

JO - Archives of Biochem and Biophysics

JF - Archives of Biochem and Biophysics

SN - 0003-9861

ER -