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Abstract
The metabolic networks in microbes and other organisms are highly complicated to ensure the robustness of the organism to adapt to different environments and to respond to internal or external stress. Several reactive species contribute to internal stress. Characterization of the highly conserved Rid protein superfamily revealed a metabolic stress caused by reactive imine/enamine species, one of which is 2-aminoacrylate (2AA), a reactive intermediate generated by pyridoxal 5’phosphate (PLP)-dependent enzymes. In Salmonella enterica that lacks RidA, 2AA accumulates and inactivates its target PLP-dependent enzymes (PLPDEs), causing perturbations that can be phenotypical in some conditions. The research that follows aims to expand the understanding of 2AA-mediated damage in S. enterica lacking a functional RidA. First, using a biochemical approach, 2AA damage in vivo to individual PLPDEs in S. enterica was quantified by detecting the PLP-pyruvate adduct formed from either a wildtype or a ridA background. Importantly, the implemented system detected 2AA damage for an IlvE enzyme from a wildtype background. This unexpected result indicated the existence of 2AA stress in the presence of a functional RidA. While the system in this work enabled assessment of 2AA damage for individual PLPDEs, the following study used a chemo-proteomic platform at the Sieber Lab from Technical University of Munich, Germany, to examine 2AA damage in S. enterica in a global context. Data from this analysis in progress will outline the S. enterica PLP-associating proteome (PLPome), and define a 2AA-damaged-PLPome by comparing PLPome from backgrounds with and without 2AA stress. On the other hand, genetic approach of the next study identified a correlation of decreased tetrahydrofolate synthesis and reduced 2AA level for a ridA strain. A model was proposed, suggesting the reduction of 2AA stress could result from higher threonine availability to the primary 2AA generator IlvA. In general, the work here presents a good example that the joint effort of biochemical approach and traditional genetic approach enables a more comprehensive view on physiological changes, and mass data analyses including proteomics powered by information technology have become increasingly helpful in providing a global perspective to help us better understand the collective events happening in cell.