Nucleotide biosynthesis is one of the vital mechanisms of living organisms. The salvage pathway of nucleotide synthesis governs an energy efficient route, utilizes the intermediates from the degradation pathway and provides the swift supply of nucleotides needed for essential cellular activities. The adenine phosphoribosyltransferase (APRT) is a part of the purine salvage pathway enzyme that catalyzes the transfer of the phosphoribosyl moiety from 5-phosphoribosyl 1-pyrophosphate (PRPP) to free nucleotide bases. Though this enzyme is well characterized across various taxonomical orders still the details of substrate specificity, enzyme activity and its physiological role in survival are still unexplored in Enterobacterales. This group comprised species that form the largest group of bacterial pathogens along with being the part of gastrointestinal tract microflora in humans. Therefore, a thorough investigation of the function of Enterobacterales's APRT and identification of a novel mechanism may aid in the development of new antibiotics to prevent the spread of multidrug resistance strains of the Enterobacterales. In order to delineate molecular mechanism of the substrate binding, we determined the high-resolution crystal structure of E. coli APRT (EcAPRT) in both apo and adenine bound forms. The EcAPRT enzyme has a core PRPP binding domain and a distinct auxiliary region comprising an N-terminal hood and flexible loop. The binding of adenine in EcAPRT is facilitated through an interesting mechanism in which the side chain conformation of carboxy-terminal residue flips and a substrate trap is generated by the side chain residues.