Protein post-translational modifications (PTMs) derived from primary metabolites have emerged as fundamental mechanisms linking cellular metabolism to physiological regulation. Here, we report the discovery and characterization of lysine fumarylation (Kfu), a previously unrecognized PTM originating from the tricarboxylic acid (TCA) cycle intermediate fumarate. Utilizing an open-search mass spectrometry approach, we identified a mass shift of +98.0002 Da on lysine residues in Escherichia coli, corresponding to the addition of a fumaryl group. By enrichment with a pan-succinyl-lysine antibody followed by mass spectrometry analysis, we demonstrated that fumarate significantly elevates global Kfu levels and mapped 857 endogenous Kfu sites. The occurrence and structural identity of Kfu were confirmed through chromatographic retention and MS/MS fragmentation comparisons with heavy isotope-labeled synthetic peptides, as well as metabolic tracing using deuterated fumarate. We further elucidate the enzymatic pathway regulating this modification: The SucC-SucD complex functions as a bona fide fumaryl-CoA synthetase, converting fumarate to fumaryl-CoA; SpeG catalyzes fumaryl group transfer to lysine substrates; and CobB acts as an NAD+-dependent defumarylase. Integrated transcriptomic and proteomic analyses suggest that Kfu regulates genes involved in stress responses, including temperature and oxidative stress pathways. This work shows lysine fumarylation as a distinct metabolic signaling mechanism, expands the repertoire of protein acylations, and provides a molecular framework for understanding how fumarate exerts its regulatory functions through covalent protein modification.
[1]Tianjin Med Univ, Dept Biochem & Mol Biol, Key Lab Immune Microenvironm & Dis, Minist Educ,Sch Basic Med Sci, Tianjin 300070, Peoples R China.
[2]Tianjin Med Univ, Key Lab Breast Canc Prevent & Therapy, Prov & Minist Cosponsored Collaborat Innovat Ctr M, Canc Inst & Hosp,Minist Educ, Tianjin 300070, Peoples R China.
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