A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry

Abstract

De novo fatty acid synthesis is a pivotal enzymatic process in all eukaryotic organisms. It is involved in the conversion of glucose and other nutrients to fatty acyl (FA) chains, that cells use as building blocks for membranes, energy storage, and signaling molecules. Central to this multistep enzymatic process is the cytosolic type I fatty acid synthase complex (FASN) which in mammals produces, according to biochemical textbooks, primarily non-esterified palmitic acid (NEFA 16:0). The activity of FASN is commonly measured using a spectrophotometry-based assay that monitors the consumption of the reactant NADPH. This assay is indirect, can be biased by interfering processes that use NADPH, and cannot report the NEFA chain-length produced by FASN. To circumvent these analytical caveats, we developed a simple mass spectrometry-based assay that affords monitoring of FASN activity and its product-specificity. In this assay (i) purified FASN is incubated with 13C-labeled malonyl-CoA, acetyl-CoA, and NADPH, (ii) at defined time points the reaction mixture is spiked with an internal NEFA standard and extracted, and (iii) the extract is analyzed directly, without vacuum evaporation and chemical derivatization, by direct-infusion high-resolution mass spectrometry in negative ion mode. This assay supports essentially noise-free detection and absolute quantification of de novo synthetized 13C-labled NEFAs. We demonstrate the efficacy of our assay by determining the specific activity of purified cow FASN and show that in addition to the canonical NEFA 16:0 this enzyme also produces NEFA 12:0, 14:0, 18:0, and 20:0. We note that our assay is generic and can be carried out using commonly available high-resolution mass spectrometers with a resolving power as low as 95,000. We deem that our simple assay could be used as high-throughput screening technology for developing potent FASN inhibitors and for enzyme engineering aimed at modulating the activity and the product-landscape of fatty acid synthases.

Fatty Acid Synthase inhibitor TVB-3166 prevents S-acylation of the Spike protein of human coronaviruses

“…6C), suggests that the FASN is also critical to generating the CoA species used for these reactions. In support of this notion, two recent studies have found that FASN generates an array of fatty acyl chains than just 16:0 and that FASN supports the generation of myristoyl-CoA and stearoyl-CoA and their subsequent attachment to proteins (61,62). Does the loss of ZDHHC5 and FASN activity impact the SARS-CoV2 infection cycle by other mechanisms?…”

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“…Recent advances in high-throughput MS resulted in highly sensitive and accurate methods for analyzing complex analytes, which have been especially useful for protein engineering applications. − By utilizing native substrates and ligands, MS is capable of performing label-free high-throughput assays for characterizing complex mixtures of engineered proteins. However, no individual MS technique provides the ability to screen all proteins in a sample with enough information to characterize all possible modifications.…”

Phosphoproteomic Analysis across the Yeast Life Cycle Reveals Control of Fatty Acyl Chain Length by Phosphorylation of the Fatty Acid Synthase Complex

“…Furthermore, to directly test whether the FAS complex in fas2 3E cells produces a higher proportion of 18:0-CoA, we next carried out an in vitro assay to profile the FAS product specificity. To this end, we prepared cytosolic fractions containing the FAS complex and measured the incorporation of 13 C 3 -labeled malonyl-CoA into de-novo-synthesized FA chains (Topolska et al, 2020). Consistent with our results in vivo, the cytosol of fas2 3E cells produced a higher proportion of 13 C-labeled FA 18:0 (Figure 6F) and yielded a 1.6-fold increased ratio between C 18and C 16 -containing FA chains as compared to wild-type cells (D-F) Summary of lipid metabolic hallmarks of indicated mutant strains cultured in YPD (left panels) and examples of lipidome differences compared to wild type (right panels).…”

“…In vitro FAS activity analysis FAS activity was assayed using 13 C 3 -malonyl-CoA and high-resolution mass spectrometry as recently described (Topolska et al, 2020). In short, BY4742 and fas2 3E cells were grown to an OD 600 of $2.5 and 50 ODu of cells were harvested (3000 g, 3 min) and washed with sterile, deionized water.…”