Purification and partial characterization of squalene epoxidase from rat liver microsomes

Ono, Teruo; Nakazono, Kiyoshi; Kosaka, Hiroko

doi:10.1016/0167-4838(82)90424-1

Cited by 80 publications

(68 citation statements)

References 34 publications

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“…However, the level of SF 86-327 required for significant inhibition of mammalian sterol synthesis is unlikely to be attained in vivo. Furthermore, squalene epoxidase, the primary target of the allylamines, is not a cytochrome P-450 enzyme (13), and so there is no likelihood of inhibiting this class of enzyme during therapy, as has been reported to occur with imidazole antimycotics…”

Section: Discussionmentioning

confidence: 99%

Specific inhibition of fungal sterol biosynthesis by SF 86-327, a new allylamine antimycotic agent

Ryder¹

1985

Antimicrob Agents Chemother

156

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SF 86-327 is a new antimycotic agent of the ailylamine type. Its primary action appears to be the inhibition of ergosterol biosynthesis at the point of squalene epoxidation, as was previously found with the related compound naftifine. Biosynthesis was measured by incorporation of [14C]acetate into sterols in cells of Candida albicans, Candida parapsilosis, Torulopsis glabrata, and the dermatophyte Trichophyton mentagrophytes. There was a positive correlation between the SF 86-327 concentrations needed for inhibition of growth and of sterol synthesis in these four fungi. The greater antifungal efficacy of SF 86-327 in comparison with naftifine was also reflected in the relative activities of the two compounds as sterol synthesis inhibitors. Inhibition was maximal at neutral pH. A similar degree of inhibition was found in cell-free extracts when [14C]mevalonate was used as substrate. In all cases, inhibition of sterol synthesis was accompanied by a parallel accumulation of labeled squalene. SF 86-327 and naftifine had no significant effect on initial enzymes of the ergosterol pathway, measured by incorporation of [14C]acetyl coenzyme A, or on steps distal to squalene epoxidation, measured by conversion of labeled squalene 2,3-epoxide or lanosterol. Both allylamines were highly selective for fungal, as opposed to mammalian, sterol biosynthesis. SF 86-327 caused slight inhibition of squalene epoxidation in a rat liver cell-free system, but at concentrations three to four orders of magnitude greater than those required for inhibition of the fungal pathway.

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Section: Discussionmentioning

confidence: 99%

Specific inhibition of fungal sterol biosynthesis by SF 86-327, a new allylamine antimycotic agent

Ryder¹

1985

Antimicrob Agents Chemother

156

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“…, squalene mono-oxygenase (4), and fatty acid elongase (5). Mice in which POR was incapacitated exhibited multiple developmental defects and embryonic lethality (6).…”

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confidence: 99%

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

McCammon

Panda

Xia

et al. 2016

Journal of Biological Chemistry

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Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo. Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.

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“…It is noteworthy that squalene epoxidase requires a flavoprotein oxidase and a NADPH-cytochrome P450 reductase (42). We, therefore, attempted to reconstitute the pepper epoxidase by incorporating stromal proteins from pepper chromoplast into the reaction mixture.…”

Section: Resultsmentioning

confidence: 99%

Xanthophyll Biosynthesis

Bouvier

d’Harlingue

Hugueney

et al. 1996

Journal of Biological Chemistry

156

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Pepper (Capsicum annuum) ␤-cyclohexenyl xanthophyll epoxidase cDNA was cloned and the corresponding enzyme overexpressed and purified from Escherichia coli, for investigation of its catalytic activity. The recombinant protein did not directly accept NADPH for epoxidation of cyclohexenyl carotenoids, nor did it operate according to a peroxygenase-based mechanism. Instead, the reducing power of NADPH was transferred to the epoxidase via reduced ferredoxin as shown by reconstitution of epoxidase activity in the presence of NADPH, ferredoxin oxidoreductase, and ferredoxin. Bacterial rubredoxin could be substituted for ferredoxin. The pepper epoxidase acted specifically on the ␤-ring of xanthophylls such as ␤-cryptoxanthin, zeaxanthin, and antheraxanthin. The proposed reaction mechanism for epoxidation involves the formation of a transient carbocation. This characteristic allows selective inhibition of the epoxidase activity by different nucleophilic diethylamine derivatives, p-dimethylaminobenzenediazonium fluoroborate and N,N-dimethyl-2-phenylaziridinium. It was also shown that the epoxidase gene was up-regulated during oxidative stress and when chloroplasts undergo differentiation into chromoplasts in pepper fruit.

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Purification and partial characterization of squalene epoxidase from rat liver microsomes

Cited by 80 publications

References 34 publications

Specific inhibition of fungal sterol biosynthesis by SF 86-327, a new allylamine antimycotic agent

Specific inhibition of fungal sterol biosynthesis by SF 86-327, a new allylamine antimycotic agent

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

Xanthophyll Biosynthesis

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