Dynamic acylome reveals metabolite driven modifications in Syntrophomonas wolfei

Fu, J. Y.; Muroski, J. M.; Arbing, M. A.; Salguero, J. A.; Wofford, N. Q.; McInerney, M. J.; Gunsalus, R. P.; Loo, J. A.; Loo, R. R. O.

doi:10.1101/2022.07.18.500474

bioRxiv · 10.1101/2022.07.18.500474

Dynamic acylome reveals metabolite driven modifications in Syntrophomonas wolfei

Fu, J. Y.·Muroski, J. M.·Arbing, M. A.·Salguero, J. A.·Wofford, N. Q.·McInerney, M. J.·Gunsalus, R. P.·Loo, J. A.·Loo, R. R. O.

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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.

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Fu, J. Y., Muroski, J. M., Arbing, M. A., Salguero, J. A., Wofford, N. Q., McInerney, M. J., Gunsalus, R. P., Loo, J. A., Loo, R. R. O.. 2022-07-19. Dynamic acylome reveals metabolite driven modifications in Syntrophomonas wolfei. https://doi.org/10.1101/2022.07.18.500474

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