Salmonella FraE, an asparaginase homolog, contributes to fructose-asparagine but not asparagine utilization.

Salmonella enterica can utilize fructose-asparagine (F-Asn) as a carbon and nitrogen source. This unusual 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 only express FraE or AnsB confirmed that each of these enzymes convert 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-P-F-Asp. Here we found that, as expected, a fraB mutant is inhibited by F-Asp as well. Collectively, these findings contribute to a better understanding of F-Asn utilization by Salmonella Importance Salmonella is so far the only organism known to be able to utilize fructose-asparagine (F-Asn) as a nutrient. We recently reported that disruption of a deglycase enzyme in the F-Asn utilization pathway inhibits the growth of Salmonella in mice, and thereby recognized that this pathway represents 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 an asparaginase activity encoded by ansB .