Deciphering allosterism of an <i>Escherichia coli</i> hexuronate metabolism regulator: UxuR

d-Glucuronate and d-galacturonate can be used by <i>Escherichia coli</i> as sole carbon sources. Their use is triggered by external environmental changes through the rearrangement of bacterial metabolic processes. Transcription factors (TFs), the key elements in tailoring gene regulation, enable environmental change responses by transcribing or repressing a gene depending on bacterial needs. Owing to its complexity, allosteric regulation remains a challenging mechanism to fully characterize. Here, the first steps of allosterism behind one TF in hexuronate metabolism in <i>E. coli</i> were revealed by combining molecular dynamics (MD) simulations, graph theory, and biosensors. Enhanced MD simulations were used to characterize one of the repressors of hexuronate metabolism, UxuR TF, in its free and ligand-bound forms. The <i>in silico</i> results provided residue selections that were tested <i>in vitro</i>. The identified residues can be divided into those that are critical for maintaining protein stability and those that are essential for facilitating allosteric communication from the effector domain to the DNA-binding site. A particularly intriguing discovery was the identification of a variant that could respond to different sugars, enriching our understanding of hexuronate metabolism flexibility. The identification of UxuR variants with altered ligand specificity not only advances our understanding of bacterial metabolism regulation but also opens new avenues for developing antimicrobial strategies targeting hexuronate pathways.