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Dynamic acylome reveals metabolite driven modifications in Syntrophomonas wolfei

ABSTRACT: Syntrophomonas wolfei is an anaerobic syntrophic microbe that degrades short-chain fatty acids to acetate, hydrogen, and/or formate. This thermodynamically unfavorable process proceeds through a series of reactive acyl-Coenzyme A species (RACS). In other prokaryotic and eukaryotic systems, the production of intrinsically reactive metabolites correlates with acyl-lysine modifications, which have been shown to play a significant role in metabolic processes. Analogous studies with syntrophic bacteria, however, are relatively unexplored and we hypothesize that highly abundant acylations could exist in S. wolfei proteins, corresponding to the RACS derived from degrading fatty acids. Here, by mass spectrometry-based proteomics (LC-MS/MS), we characterize and compare acylome profiles of two S. wolfei subspecies grown on different carbon substrates. Because modified S. wolfei proteins are sufficiently abundant for post-translational modification (PTM) analyses without antibody enrichment, we could identify types of acylations comprehensively, observing six types (acetyl-, butyryl-, 3-hydroxybutyryl-, crotonyl-, valeryl-, hexanyl-lysine), two of which have not been reported in any system previously. All of the acyl-PTMs identified correspond directly to RACS in fatty acid degradation pathways. A total of 369 sites of modification were identified on 237 proteins. Changing the carbon substrate altered the acylation profile. Moreover, structural studies and in vitro acylation assays of a heavily modified enzyme, acetyl-CoA transferase, provided insight on the possible impact of these acyl-protein modifications. Our findings link protein acylation by RACS to shifts in cellular metabolism.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Syntrophomonas Wolfei Subsp. Wolfei Str. Goettingen (ncbitaxon:335541) Syntrophomonas Wolfei Subsp. Methylbutyratica (ncbitaxon:378794)

SUBMITTER: Joseph A. Loo

PROVIDER: MSV000089730 | MassIVE | Sat Jun 25 18:39:00 BST 2022

SECONDARY ACCESSION(S): PXD034881

REPOSITORIES: MassIVE

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Project description:Fatty acid synthesis is closely linked to nutrient availability and cellular energetic status. The committed step in fatty acid synthesis is the acetyl CoA carboxylase. Eukaryotes have two genes encoding acetyl CoA carboxylases, one encoding a cytosolic enzyme and another coding for a mitochondrial enzyme. They catalyze the synthesis of malonyl CoA in the cytosol and the mitochondria, respectively. While cytosolic malonyl CoA is the precursor for fatty acid synthesis, mitochondrial malonyl CoA controls the transfer of fatty acyl group into the mitochondria by inhibiting carnitine/palmitoyl transferase activity and thus, regulates Î²-oxidation. In Saccharomyces cerevisiae, Î²-oxidation is restricted to the peroxisomes, raising the question of the function of the mitochondrial isoform (HFA1). In this study, we replaced the cytosolic Acc1 with Hfa1 expressed in the cytosol by removing the mitochondrial leader peptide, under control of the HFA1 promoter. We studied fatty acid synthesis and transcription profiles in this strain during starvation for carbon or nitrogen, using glucose or ethanol as the carbon source. Under all the conditions studied, the key sensor of energetic status, Snf1, was activated, indicating active inhibition of fatty acid synthesis. The pool size of fatty acids was smaller when Acc1 was replaced with truncated Hfa1 for fatty acid synthesis. Yet, the transcription profiles were similar in both the cases. These results point towards the conclusion that Hfa1 is either catalytically less efficient or it is more sensitive to inhibition by Snf1. Gene expression from a strain of Saccharomyces cerevisiae where cytosolic fatty acid synthesis occurs by mitochondrial acetyl CoA carboxylase (without its mitochondrial leader peptide) is compared with that in a reference strain while growing in chemostats on carbon or nitrogen starvation using glucose or ethanol as the carbon source. There are two strains (reference or mutant), two carbon sources (glucose or ethanol) and two limitations (carbon or nitrogen), resulting in 8 comparisons. Each array was performed in duplicate, resulting in 16 CEL files. Growth was limited by either carbon or nitrogen. When carbon was the limited nutrient, we tested growth on either glucose or ethanol (both using ammonium sulfate as the nitrogen source). When ammonium sulfate was limiting, we used either glucose or ethanol as the carbon source.

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Dynamic acylome reveals metabolite driven modifications in Syntrophomonas wolfei

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