Discovery and Enzymatic Regulation of Lysine Fumarylation, a Post-Translational Modification in Bacteria

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.