In bacteria, the biosynthetic pathway for the hydroxymethyl pyrimidine moiety of thiamine shares metabolic intermediates with purine biosynthesis. The two pathways branch after the compound aminoimidazole ribotide. Past work has shown that the first common metabolite, phosphoribosyl amine (PRA), can be generated in the absence of the first enzyme in purine biosynthesis, PurF. PurF-independent PRA synthesis is dependent on both strain background and growth conditions. Standard genetic approaches have not identified a gene product singly responsible for PurF-independent PRA formation. This result has led to the hypothesis that multiple enzymes contribute to PRA synthesis, possibly as the result of side products from their dedicated reaction. A mutation that was able to restore PRA synthesis in a purF gnd mutant strain was identified and found to map in the gene coding for the TrpD subunit of the anthranilate synthase (AS)-phosphoribosyl transferase (PRT) complex. Genetic analyses indicated that wild-type AS-PRT was able to generate PRA in vivo and that the P362L mutant of TrpD facilitated this synthesis. In vitro activity assays showed that the mutant AS was able to generate PRA from ammonia and phosphoribosyl pyrophosphate. This work identifies a new reaction catalyzed by AS-PRT and considers it in the context of cellular thiamine synthesis and metabolic flexibility.Metabolic processes in a living cell are connected through the integration of biochemical pathways. This metabolic integration allows bacteria to survive under diverse conditions and is essential for their adaptation to changing environments. The structure and function of networks created by the integration and overlap of metabolic processes are largely uncharacterized. The thiamine biosynthetic pathway in Salmonella enterica has direct biochemical connections with other metabolic pathways, including purine and isoprenoid biosynthesis (5,33,40), and indirect connections with a number of other cellular processes (1,16,19,25,36). Thus, this biosynthetic pathway serves as a focal point for studies to identify and characterize metabolic interactions, redundancy, and regulation that are essential in a robust metabolism.