Salmonella enterica can utilize fructose-asparagine (F-Asn) as a source of carbon and nitrogen. This capability has been attributed to five genes in the fra locus. Previously, we determined that mutations in fraB (deglycase), fraD (kinase), or fraA (transporter) eliminated the ability of Salmonella to grow on F-Asn, while a mutation in fraE allowed partial growth. We hypothesized that FraE, a putative periplasmic fructose-asparaginase, converts F-Asn to NH₄ ⁺ and fructose-aspartate (F-Asp). FraA could then transport F-Asp into the cytoplasm for subsequent catabolism. Here, we report that growth of the fraE mutant on F-Asn is caused by a partially redundant activity provided by AnsB, a periplasmic asparaginase. Indeed, a fraE ansB double mutant is unable to grow on F-Asn. Moreover, biochemical assays using periplasmic extracts of mutants that express only FraE or AnsB confirmed that each of these enzymes converts F-Asn to F-Asp and NH₄ ⁺ However, FraE does not contribute to growth on asparagine. We tested and confirmed the hypothesis that a fraE ansB mutant can grow on F-Asp, while mutants lacking fraA, fraD, or fraB cannot. This finding provides strong evidence that FraA transports F-Asp but not F-Asn from the periplasm to the cytoplasm. Previously, we determined that F-Asn is toxic to a fraB mutant due to the accumulation of the FraB substrate, 6-phosphofructose-aspartate (6-P-F-Asp). Here, we found that, as expected, a fraB mutant is also inhibited by F-Asp. Collectively, these findings contribute to a better understanding of F-Asn utilization by Salmonella IMPORTANCE Salmonella is able to utilize fructose-asparagine (F-Asn) as a nutrient. We recently reported that the disruption of a deglycase enzyme in the F-Asn utilization pathway inhibits the growth of Salmonella in mice and recognized this pathway as a novel and specific drug target. Here, we characterize the first step in the pathway wherein FraE hydrolyzes F-Asn to release NH₄ ⁺ and F-Asp in the periplasm of the cell. A fraE mutant continues to grow slowly on F-Asn due to asparaginase activity encoded by ansB.

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