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Microsomal enzymes of cholesterol biosynthesis from lanosterol: A progress report
Author(s) -
Trazaskos James M.,
Bowen Wayne D.,
Fisher Gary J.,
Billheimer Jeffrey T.,
Gaylor James L.
Publication year - 1982
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/bf02535112
Subject(s) - biochemistry , lanosterol , chemistry , reductase , cytochrome , enzyme , cytochrome p450 reductase , microsome , demethylation , sterol , cytochrome c , coenzyme q – cytochrome c reductase , cholesterol , mitochondrion , gene expression , dna methylation , gene
Abstract Our principal goal is the complete resolution and reconstitution of the microsomal enzymes of cholesterol biosynthesis. Elucidation of the enzymology has been achieved primarily through dissection of the membrane‐bound, 19‐step multienzymic process. This report describes the dissection approach through both interruption of specific steps and reconstitution of enzymes that catalyze oxidation of the 14α‐methyl group. In earlier work, 4‐demethylation was resolved into 3 component reactions catalyzed by: 4‐methyl sterol oxidase (NAD[P] H‐ and O 2 ‐dependnet); steroid 4α‐carboxylic acid decarboxylase (NAD‐dependent); and 3‐ketosteroid reductase (NADPH‐dependent). The 3‐ketosteroid reductase and decarboxylase have been solubilized with Lubrol WX and deoxycholate, respectively, and characterized. The 4‐methyl sterol oxidase (cytochrome b 5 ‐dependent) recently has been solubilized with Renex 690. This study represents successful elucidation of a microsomal enzyme sequence by interruption of the central 10‐step segment of the multienzymic formation of cholesterol from lanosterol. The initial C‐32 oxidative reaction of 14α‐methyl group elimination is catalyzed by a from of cytochrome P‐450 that is induced by isosafrole. The induced cytochrome P‐450 has been solubilized with Emulgen 913 and purified to homogeneity (17 nmol of cytochrome/mg protein). 24,25‐Dihydrolanosterol is oxidized by combination of cytochrome P‐450 reductase, hematin, NADPH, glutathione, and the purified, isosafrole‐induced cytochrome in an artificial liposome. Oxidation product identification is underway. This study represents successful elucidation of a microsomal multienzymic sequence by solubilization and reconstitution of a segment of the pathway. The remaining enzymes under study are the Δ 8 →Δ 7 isomerase and 3 NADPH‐dependent double bond reductases that catalyze reduction of: Δ 7 , Δ 14 ‐ Δ 24 ‐sterol double bonds. Purification of these nonoxygenrequiring enzymes is in progress. Resolution of the enzymes has demonstrated unequivocally that cholesterol synthesis via this pathway could not have appeared biologically until membranes contained both the cytochrome P‐450‐ and cytochrome b 5 ‐electron transport enzymes. Chemically, all enzymic attacks in the formation of cholesterol from lanosterol appear to be initiated on the α‐face of the relatively planar steroids. Thus, considerable genetic pressure must have been needed for the stereospecific clearing of the steroidal α‐face to form the mature membrane component, cholesterol.