Reactive metabolites are produced by many biochemical pathways within a given metabolic network. The inherent reactivity of these metabolites presents a challenge to cells, where integrated biochemical pathways often coexist in densely packed spaces, paving the way for aberrant metabolic interactions to arise. Many networks produce reactive metabolite degradation systems to prevent these labile metabolites from damaging the cell. The broadly conserved Rid protein superfamily represents a key line of defense against reactive enamine stress. Reactive enamines, specifically 2-aminoacrylate generated by pyridoxal 5-phophate-dependent serine/threonine dehydratases, accumulate in Salmonella enterica lacking its native enamine deaminase, RidA. Persistence of 2-aminoacrylate in the absence of RidA triggers the inactivation of several distinct pyridoxal 5-phophate-dependent enzymes, diminishing cell fitness. The research described herein was performed to characterize diversity in the mechanisms of 2-aminoacrylate production, to improve our understanding of the key growth-limiting metabolic consequence of 2-aminoacrylate stress in S. enterica, and to assess conservation of the RidA-2-aminoacrylate paradigm in eukaryotic organisms. The data show that multiple mechanisms of 2-aminoacrylate production are found in S. enterica, the primary growth-limiting defect caused by 2-aminoacrylate stress in S. enterica ridA mutant strains is diminished glycine/one-carbon unit biosynthesis, and the RidA paradigm for reactive enamine control is conserved in yeast, where disruption of the mitochondrial RidA homolog (Mmf1p) leads to 2-aminoacrylate-induced loss of the mitochondrial genome.