Effect of ay-9944 on sterol biosynthesis in Chlorella sorokiniana

doi:10.1016/S0031-9422(00)88842-1

Phytochemistry

Volume 15, Issue 12, 1976, Pages 1907-1910

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Abstract

When Chlorella sorokiniana was grown in the presence of 4 ppm AY-9944 total sterol production was unaltered in comparison to control cultures. However, inhibition of sterol biosynthesis was shown by the accumulation of a number of sterols which were considered to be intermediates in sterol biosynthesis. The sterols which were found in treated cultures were identified as cyclolaudenol, 4α,14α-dimethyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 4α,14α-dimethyl -5α-ergosta-8,25-dien-3β-ol, 14α-methyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 24-methylpollinastanol, 14α-methyl-5α-ergost-8-en-3β-ol, 5α-ergost -8(14)-enol, 5α-ergost-8-enol, 5α-ergosta-8(14),22-dienol, 5α-ergosta-8,22-dienol, 5α-ergosta-8,14-dienol, and 5α-ergosta-7,22-dienol, in addition to the normally occurring sterols which are ergosterol, 5α-ergost-7-enol, and ergosta-5,7-dienol.

The occurrence of these sterols in the treated culture indicates that AY-9944 is an effective inhibitor of the Δ⁸ → Δ⁷ isomerase and Δ¹⁴-reductase, and also inhibits introduction of the Δ²²-double bond. The occurrence of 14α-dimethyl-5α-ergosta-8,25-dien-3β-ol and 14α-methyl-9β,19-cyclo-5α-ergost -25-en-3β-ol is reported for the first time in living organisms. The presence of 25-methylene sterols suggests that they, and not 24-methylene derivatives, are intermediates in the biosynthesis of sterols in C. sorokiniana.

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Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the Δ⁸–Δ⁷ isomerase (Erg2p) or Δ⁵ desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5α-cholest-8-en-3β-ol, 8-dehydrocholesterol (Δ^5,8 sterol), or isodehydrocholesterol (Δ^6,8 sterol), together with the corresponding $3 α -^{3} H$ isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven Δ^22E sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag⁺-HPLC) and identified by gas chromatography–mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag⁺-HPLC. When Δ⁸–Δ⁷ isomerization was blocked, exogenous Δ⁸ sterol underwent desaturation to Δ^5,8, Δ^6,8, and Δ^8,14 sterols. Formation of Δ^5,8 sterol was strongly favored over Δ^6,8 sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The Δ^5,8 sterol was metabolically almost inert except for Δ²² desaturation, whereas the Δ^6,8 sterol was readily converted to Δ^5,7, Δ^5,7,9(11), and Δ^7,9(11) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, Δ^5,7 sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of Δ^5,8 and Δ^5,7,9(11) sterols in wild-type yeast compared to Smith–Lemli–Opitz individuals.
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Azasqualenes, bis-azasqualenes and derivatives, designed as inhibitors of squalene 2,3-epoxide cyclase, a key enzyme in sterol biosynthesis, were synthesized and their in vitro activities against a variety of yeasts, fungi, gram-positive and gram-negative bacteria were determined.
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[6-³H₁] (24S)-24-Ethylcholesta-5,22,25-trien-3β-ol added to the growth medium of a culture of Trebouxia sp. 213/3 was efficiently taken-up by the cells and converted into (24R)-24-ethylcholesta-5,22-dien-3β-ol (poriferasterol) which is one of the major sterols of this alga. A cell-free homogenate was obtained from Trebouxia which catalysed the NADPH-dependent reduction of [6-³H₁] (24S)-24-ethylcholesta-5,22,25-trien-3β-ol to yield poriferasterol. The δ²⁵-sterol reductase was found to be mainly localized in the microsomal fraction of the homogenate.
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The concentration of triarimol giving ca 50% inhibition of growth was different for each of 3 species of Chlorella [C. emersonii, 1 mg/l. (1.5 × 10⁻⁶ M), C. ellipsoidea 10 mg/l. (3 × 10⁻⁵ M), C. sorokiniana, 2 mg/l. (6 × 10⁻⁶ M)]. The total lipid of 3 species of Chlorella grown in a culture medium containing triarimol were analysed for chlorophyll, fatty acids and sterol composition. Growth rates were studied in the presence of different concentrations of triarimol. The growth rates of the 3 species were differentially inhibited by triarimol. The growth of Chlorella sorokiniana was 50% inhibited by 2 mg/l. triarimol but 20 mg/l. did not produce a cessation of growth. The greatest inhibition of growth rates and chlorophyll content was observed in Chlorella emersonii. The quantity of unsaturated fatty acids was increased by triarimol treatment in all 3 species of Chlorella. Triarimol strongly inhibited 14α-demethylation in Chlorella emersonii, and C. ellipsoidea and less in C. sorokiniana, resulting in accumulation of 14α-methyl sterols. Triarimol also inhibited the second alkylation of the side chain in C. ellipsoidea and C. emersonii. The introduction of the 22-double bond was inhibited in all 3 species of Chlorella studied. Although some differences were apparent, the effect of triarimol was quite similar to that of triparanol and AY-9944 in these 3 species of Chlorella.
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^☆: Scientific Article No. A2193 Contribution No. 5168 of the Marryland Agricultural Experiment Station.

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Effect of ay-9944 on sterol biosynthesis in Chlorella sorokiniana☆

Abstract

J. Lipid Res.

Biochem. Biophys. Res. Commun.

Phytochemistry

J. Lipid Res.

Nature

Biochemistry

Proc. Roy. Soc. London B.

Lipids

Lipids

Lipids