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. 2015 Mar 31;112(13):3955-60.
doi: 10.1073/pnas.1423951112. Epub 2015 Mar 16.

Acyl-CoA oxidase complexes control the chemical message produced by Caenorhabditis elegans

Xinxing Zhang  1 , Likui Feng  1 , Satya Chinta  1 , Prashant Singh  1 , Yuting Wang  1 , Joshawna K Nunnery  1 , Rebecca A Butcher  2
Affiliations

Acyl-CoA oxidase complexes control the chemical message produced by Caenorhabditis elegans

Xinxing Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

Caenorhabditis elegans uses ascaroside pheromones to induce development of the stress-resistant dauer larval stage and to coordinate various behaviors. Peroxisomal β-oxidation cycles are required for the biosynthesis of the fatty acid-derived side chains of the ascarosides. Here we show that three acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, form different protein homo- and heterodimers with distinct substrate preferences. Mutations in the acyl-CoA oxidase genes acox-1, -2, and -3 led to specific defects in ascaroside production. When the acyl-CoA oxidases were expressed alone or in pairs and purified, the resulting acyl-CoA oxidase homo- and heterodimers displayed different side-chain length preferences in an in vitro activity assay. Specifically, an ACOX-1 homodimer controls the production of ascarosides with side chains with nine or fewer carbons, an ACOX-1/ACOX-3 heterodimer controls the production of those with side chains with seven or fewer carbons, and an ACOX-2 homodimer controls the production of those with ω-side chains with less than five carbons. Our results support a biosynthetic model in which β-oxidation enzymes act directly on the CoA-thioesters of ascaroside biosynthetic precursors. Furthermore, we identify environmental conditions, including high temperature and low food availability, that induce the expression of acox-2 and/or acox-3 and lead to corresponding changes in ascaroside production. Thus, our work uncovers an important mechanism by which C. elegans increases the production of the most potent dauer pheromones, those with the shortest side chains, under specific environmental conditions.

Keywords: acyl-CoA oxidase; ascaroside; beta-oxidation; dauer; pheromone.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Ascaroside pheromones and β-oxidation enzymes implicated in their biosynthesis. (A) Dauer-inducing ascaroside pheromones. (B) Modular structure of the ascarosides. The ascarosides can be named according to the number of carbons in their side chains using the following rubric: head group-asc-(ω)(Δ)C#-terminus group. Head groups can be attached via the 4′-hydroxyl of ascarylose (e.g., indole-3-carbonyl group) or the 2′-hydroxyl of ascarylose (e.g., glucosyl group). Modifications to the terminus of the side chain include para-aminobenzoic acid and methylketone. R = H for the ω-ascarosides and R = CH3 for the (ω-1)-ascarosides. (C) The β-oxidation enzymes implicated in ascaroside biosynthesis. The red dot tracks the position of the β-carbon during the β-oxidation cycle.
Fig. 2.
Fig. 2.
LC-MS/MS analysis of the ascarosides produced by acyl-CoA oxidase mutants. The ascarosides produced by the acox-1 deletion mutant (A), acox-3 deletion mutant (B), and acox-2 nonsense mutant (C). Data represent the average of at least two experiments ± SD.
Fig. 3.
Fig. 3.
In vitro activity of acyl-CoA oxidase homo- and heterodimers. (A) Synthetic substrates, including CoA-thioesters of ascarosides, fatty acids, and hydroxylated fatty acids. (B–D) Activity of the ACOX-1 homodimer (B), ACOX-1/ACOX-3 heterodimer (C), and ACOX-2 homodimer (D) against the substrates, tested at 30 μM. Data represent the average of three experiments ± SD. AU, absorbance units.
Fig. 4.
Fig. 4.
Ascaroside production (A and C) and expression levels of acyl-CoA oxidase genes (B and D) under different conditions. (A) Ratio of the ascarosides produced over a short time period (20 h) by fed versus nonfed L4 worms. (B) Expression of acox-1, -2, and -3 in fed versus nonfed L4 worms, 4 h after treatment. (C) Ratio of ascarosides produced by synchronized worms cultivated in non-DI versus DI conditions during the first 28 h of the time course (see Fig. S7 for the full dataset). (D) Expression of acox-1, -2, and -3 in synchronized worms cultivated in non-DI versus DI conditions at the 21-h time point. Data represent the average of two experiments ± SD.
Fig. 5.
Fig. 5.
Model for the role of specific acyl-CoA oxidase homo- and heterodimers in the biosynthetic pathway of (ω-1)-ascarosides (A) and ω-ascarosides (B). Dauer pheromone ascarosides are boxed. Dotted arrows and enzymes that appear in gray indicate hypothetical steps and activities that have not yet been verified experimentally.
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