Regulation of rat hepatic 3β-hydroxysterol Δ7-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation

S Shefer,G Salen,A Honda,A.K Batta,L.B Nguyen,G.S Tint,Y.A Ioannou,R Desnick

doi:10.1016/s0022-2275(20)33327-7

Abstract

The mechanism for the catalytic reduction of the double bond at C-7, 8 in 7-dehydrocholesterol by 3beta-hydroxysterol Delta7-reductase was investigated by testing structurally related sterols as substrates and potential inhibitors. The hepatic smooth endoplasmic reticulum was identified as the site of enzyme activity. All putative substrates contained 27 carbons, but differed from 7-dehydrocholesterol by the addition of either an ethyl substituent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl substituent at C-24 (ergosterol), epimerization of the hydroxyl from the 3beta- to 3alpha-configuration (7-dehydroepicholesterol), or a saturated double bond at C-5,6 (lathosterol). Two non-steroidal compounds that inhibit 3beta-hydroxysterol Delta7-reductase in vivo (AY 9944 and BM 15.766) were also tested. Ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol were reduced at C-7, 8 to form brassicasterol, sitosterol, and epicholesterol, respectively, but 75% less efficiently than 7-dehydrocholesterol. Increasing concentrations of these sterols competitively inhibited 3beta-hydroxysterol Delta7-reductase activity. The double bond at C-7,8 in lathosterol was not reduced. AY 9944 and BM 15.766 inhibited 3beta-hydroxysterol Delta7-reductase activity non-competitively. 3beta-Hydroxysterol-Delta7-reductase activity declined after microsomes were exposed to alkaline phosphatase, and enzyme activity was increased by phosphorylation with Mg2+, and ATP. These results demonstrate that the reduction of the double bond at C-7,8 requires binding of the enzyme protein with the B-ring of the sterol substrate that contains a double bond at C-5,6. The reaction is hindered by substituents located on the apolar side-chain and epimerization of the hydroxyl group in ring A to a 3alpha-configuration. 3beta-Hydroxysterol Delta7-reductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form.

Highlights

The mechanism for the catalytic reduction of the double bond at C-7,8 in 7-dehydrocholesterol by 3␤-hydroxysterol ⌬7-reductase was investigated by testing structurally related sterols as substrates and potential inhibitors
To better understand the enzymatic defect in the SmithLemli-Opitz syndrome, where 3␤-hydroxysterol ⌬7-reductase activity is markedly inhibited [2], we examined the mechanism for the reduction of the double bond at C-7,8 as catalyzed by 3␤-hydroxysterol ⌬7-reductase
Proof of purity was based on the recovery of cholesterol 7␣-hydroxylase activity in the various fractions. 3␤Hydroxysterol ⌬7-reductase was most active in the microsomal fraction

Summary

Introduction

The mechanism for the catalytic reduction of the double bond at C-7,8 in 7-dehydrocholesterol by 3␤-hydroxysterol ⌬7-reductase was investigated by testing structurally related sterols as substrates and potential inhibitors. Ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol were reduced at C-7,8 to form brassicasterol, sitosterol, and epicholesterol, respectively, but 75% less efficiently than 7-dehydrocholesterol Increasing concentrations of these sterols competitively inhibited 3␤-hydroxysterol ⌬7-reductase activity. 3␤-Hydroxysterol ⌬7-reductase catalyzes the last reaction in the cholesterol biosynthetic pathway, the reduction of the double bond at C-7,8 in 7-dehydrocholesterol to form cholesterol (Fig. 1). This enzyme, known as 7dehydrocholesterol ⌬7-reductase, is inherited defectively in the Smith-Lemli-Opitz syndrome, a recessive birth defect [1, 2]. A major aim was to gain insight into the ⌬7-reduction mechanism and its regulation to better understand gene mutations that might be responsible for the Smith-Lemli-Opitz syndrome

Methods

Results

Conclusion