Abstract
Acyl carrier proteins (ACPs) are the scaffolds for fatty acid biosynthesis in living systems, rendering them essential to a comprehensive understanding of lipid metabolism. However, accurate quantitative methods to assess individual acyl-ACPs do not exist. We developed a robust method to quantify acyl-ACPs to the picogram level. We successfully identified acyl-ACP elongation intermediates (3-hydroxyacyl-ACPs and 2,3-trans-enoyl-ACPs) and unexpected medium-chain (C10:1, C14:1) and polyunsaturated long-chain (C16:3) acyl-ACPs, indicating both the sensitivity of the method and how current descriptions of lipid metabolism and ACP function are incomplete. Such ACPs are likely important to medium-chain lipid production for fuels and highlight poorly understood lipid remodeling events in the chloroplast. The approach is broadly applicable to type II fatty acid synthase systems found in plants and bacteria as well as mitochondria from mammals and fungi because it capitalizes on a highly conserved Asp-Ser-Leu-Asp amino acid sequence in ACPs to which acyl groups attach. Our method allows for sensitive quantification using liquid chromatography-tandem mass spectrometry with de novo-generated standards and an isotopic dilution strategy and will fill a gap in our understanding, providing insights through quantitative exploration of fatty acid biosynthesis processes for optimal biofuels, renewable feedstocks, and medical studies in health and disease.
Highlights
The synthesis of fatty acyl chains is essential for the production of storage and membrane lipids as well as functional molecules that modulate gene expression or contribute to protein activity
Carbon chains of up to 18 carbons in length are produced during fatty acid biosynthesis (Li-Beisson et al, 2013) on an acyl carrier protein scaffold (Overath and Stumpf, 1964) that is connected to the acyl chain through linkage of a 49phosphopantetheine group to a Ser residue found in the acyl carrier protein (ACP; Figure 1A)
Peptide hydrolysis at aspartate residues produces an acyl molecular species that can be processed for characteristic fragments by mass spectrometry that have precursor-product losses of 315.1 and 413.1 D. (B) ACPs are central to fatty acid synthesis with the process of fatty acid biosynthetic elongation of an acyl chain by two carbons taking place on ACP intermediates through a series of enzymatic steps that are repeated and conclude with acyl transfer to glycerol-3-phosphate or lyso-phosphatidic acid for production of chloroplast lipids or hydrolysis by a thioesterase
Summary
The synthesis of fatty acyl chains is essential for the production of storage and membrane lipids as well as functional molecules that modulate gene expression or contribute to protein activity. The chain elongation process is terminated in the chloroplast by acylACP–dependent acyltransferases or by thioesterase reactions; the former contribute acyl groups for lipid biosynthesis in the chloroplast, while the latter release a nonesterified fatty acid from the ACP that can be later exported. In both cases, the ACP substrate pool is regenerated for further fatty acid biosynthetic reactions. Nonesterified fatty acids can be activated to acyl-CoA molecules (acyl-CoAs) outside the chloroplast to make glycerolipids for storage oil production in the endoplasmic reticulum, waxes, surface lipids, or membrane biogenesis (Li-Beisson et al, 2013)
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