Abstract
Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ(24(28))-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphate not present in fungi. Here, we report that C. reinhardtii synthesizes the protosterol cycloartenol and converts it to ergosterol (C24β-methyl) and 7-dehydroporiferasterol (C24β-ethyl) through a highly conserved sterol C24- methylation-C25-reduction (Δ(25(27))-olefin) pathway that is distinct from the well-described acetate-mevalonate pathway to fungal lanosterol and its conversion to ergosterol by the Δ(24(28))-olefin pathway. We isolated and characterized 23 sterols by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol-treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24β-methyl/ethyl groups and Δ(22(23))E and Δ(25(27))-double bond constructions. When incubated with [methyl-(2)H(3)]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24β-alkyl sterols, consistent only with a Δ(25(27))-olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol.
Highlights
Ergosterol is the predominant sterol of fungi and green algae
The addition of C1 to C3 side chains is derived by transmethylation reactions requiring S-adenosyl-L-methionine as the methyl donor and catalyzed by the sterol C24-methyltransferase (24-SMT) family of enzymes (1, 2)
A striking finding to emerge from in vivo isotopically labeling, sterol biosynthesis inhibitor-treatments, and cell-free 24-SMT enzyme studies among phylogenetically diverse algae that include the green, brown, and golden brown algae (15–18) that is not necessarily obvious by current bioinformatic analyses (19) is that ergosterol biosynthesis in the green algae may use a different set of enzymatic reactions in module III from fungi
Summary
C. reinhardtii strains and culture conditions C. reinhardtii wild-type strains 21gr (mt+; CC-1690 and 6145c; CC-1691) and ergosterol mutants KD7 and KD21 (24), obtained from the Chlamydomonas Genetics Center, Duke University (Durham, NC), were grown at 23°C on a 13:11 h light:dark cycle with aeration in medium I or medium II of Sager and Granick (25) as previously described (26). Cells (1 × 106/ml) were cultured for 3 days in medium I containing 1 μM 25-thialanosterol iodide salt. Feeding of [methyl-2H3]methionine to C. reinhardtii L-[methyl-2H3]methionine (98 atom % of 2H) (Sigma, St. Louis) was administered to wild-type C. reinhardtii cultures inoculated with 1 × 107 cells/ml at 1 mg/ml. After 3 days inoculation under light, the cells were harvested by centrifugation, and the total sterols from the cultures were examined by GC-MS
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