Amadori products are stable sugar‐amino acid conjugates that are formed non‐enzymatically via the Maillard reaction that takes place during preparation of foods by heating, roasting, and drying. Fructose‐lysine (F‐Lys, ε‐conjugated) is one of the most abundant Amadori compounds in processed foods and is a key intermediate in the formation of advanced glycation end products, which in turn are implicated in inflammation and disease. The variation among humans in their ability to metabolize F‐Lys has motivated an examination of the inter‐individual differences in gut microbial taxa and the enzymes that help convert F‐Lys into short‐chain fatty acids or cellular energy. Results from such studies are also expected to yield insights into whether F‐Lys utilization by bacterial pathogens (e.g., Escherichia coli, Salmonella enterica serovar Typhimurium) might offer them a competitive edge. Either during or after bacterial uptake, F‐Lys is phosphorylated to form 6‐phosphofructose‐lysine (6‐P‐F‐Lys). FrlB, a deglycase, converts 6‐P‐F‐Lys to L‐lysine and glucose 6‐phosphate, with the latter feeding into glycolysis. Since the catalytic mechanism of FrlB has not been studied, we sought to obtain a high‐resolution structure of Salmonella FrlB ± 6‐P‐F‐Lys and identify the active‐site residues essential for catalysis. After overexpression and purification of recombinant Salmonella FrlB, we obtained its 1.9 Å crystal structure. FrlB exists as a dimer with two identifiable inter‐subunit active sites. In the absence of a co‐crystal structure of FrlB with 6‐P‐F‐Lys, we took two different strategies to delineate its catalytic pocket. First, we observed that the phosphosugar‐binding module–called the sugar isomerase (SIS) domain in FrlB–shared sequence similarity with the eponymous domain in E. coli glucosamine 6‐phosphate synthase (GlmS), which generates glucosamine 6‐phosphate from fructose 6‐phosphate (F‐6‐P) and glutamine. Overlaying the tertiary structures of Salmonella FrlB with E. coli GlmS, which had previously been co‐crystallized with F‐6‐P, helped identify FrlB residues that could be involved in 6‐P‐F‐Lys binding and cleavage. Second, sequence alignment of Salmonella FrlB with FraB, a related and biochemically characterized deglycase required for metabolism of fructose‐asparagine (an Amadori compound) pinpointed residues that could act as a general acid and a general base during deglycation. From these comparative analyses, six candidate residues in FrlB were individually mutated to alanine or another conservative substitution, and the mutant derivatives were purified using affinity chromatography. Our differential scanning fluorimetry studies revealed that all the mutants exhibit thermal stability nearly identical to wild‐type FrlB; importantly, our native mass spectrometry (nMS) studies confirmed that the mutations did not impair the ability of these mutants to form a stable dimer. A spectrophotometric coupled assay was employed to measure the activity of FrlB and the panel of mutants. When a mutation dampened or eliminate...

Although salmonellosis, an infectious disease, is a significant global healthcare burden, there are no Salmonella-specific vaccines or therapeutics for humans. Motivated by our finding that FraB, a Salmonella deglycase responsible for fructose-asparagine catabolism, is a viable drug target, we initiated experimental and computational efforts to identify inhibitors of FraB. To this end, our recent high-throughput screening initiative yielded almost exclusively uncompetitive inhibitors of FraB. In parallel with this advance, we report here how a separate structural and computational biology investigation of FrlB, a FraB paralog, led to the serendipitous discovery that 2-deoxy-6-phosphogluconate is a competitive inhibitor of FraB (KI ~ 3 μM). However, this compound was ineffective in inhibiting the growth of Salmonella in a liquid culture. In addition to poor uptake, cellular metabolic transformations by a Salmonella dehydrogenase and different phosphatases likely undermined the efficacy of 2-deoxy-6-phosphogluconate in live-cell assays. These insights inform our ongoing efforts to synthesize non-hydrolyzable/-metabolizable analogs of 2-deoxy-6-phosphogluconate. We showcase our findings largely to (re)emphasize the role of serendipity and the importance of multi-pronged approaches in drug discovery.

The foodborne pathogen Salmonella enterica serovar Typhimurim (Salmonella) causes approximately 94 million enteric infections and 50,000 diarrheal deaths annually worldwide. The Center for Disease Control and Prevention estimates 1.35 million Salmonella‐related illnesses in the United States annually. There are no vaccines or antibiotics to specifically combat this bacterium. During inflammation post‐infection, Salmonella exploits fructose‐asparagine (F‐Asn) as a carbon and nitrogen source. F‐Asn–formed during cooking or dehydration of raw foods–is a product of an Amadori rearrangement following the non‐enzymatic condensation of glucose and asparagine. F‐Asn is metabolized using three enzymes and a transporter encoded by the fra operon. The roles of a periplasmic asparaginase (FraE), cytoplasmic kinase (FraD), and a cytoplasmic deglycase (FraB) in F‐Asn catabolism are now established. Importantly, the FraB deglycase was identified as a promising drug target because FraB dysfunction led to accumulation of its substrate and self‐poisoning of Salmonella. The current work was undertaken to characterize gene regulation of the fra operon by FraR, the putative transcription factor in this locus. FraR is predicted to be a member of the GntR transcription factor family with an N‐terminal DNA‐binding domain (DBD) and a C‐terminal inducer‐binding domain (IBD). We hypothesize that FraR binds to the fraB promoter in vivo and acts as a transcriptional repressor, and that binding of an inducer to the FraR‐IBD triggers a conformational change to release the DNA from the DBD and thereby permit transcription of the fra genes. This postulate was tested by first purifying recombinant FraR (post‐overexpression in Escherichia coli), and then assessing FraR‐DNA binding affinity (± putative inducers) using two cross‐validating methods: fluorescence‐based gel‐shift assays and online buffer exchange (OBE) coupled to native mass spectrometry (nMS). With OBE, samples are kept in a non‐volatile buffer that favors their native biological properties and then buffer exchanged into ammonium acetate on‐line for MS analysis. Thus, OBE‐nMS eliminates difficulties typically associated with sample preparation and expedites characterization of protein and protein‐DNA complexes. Here, we used OBE‐nMS to confirm the oligomeric state of FraR and FraR‐DNA complexes and to investigate 6‐phospho‐fructose‐aspartate (6‐P‐F‐Asp) as a potential inducer. Results from our studies have provided insights into the binding stoichiometry of FraR, operator sequence, and inducer identity. We showed that a FraR dimer binds with high affinity to two 26‐bp DNA fragments (KD ~1 nM), and that two 6‐P‐F‐Asp molecules bind to each dimer (KD ~2 µM). Our studies have also shown that 6‐P‐F‐Asp acts as the inducer that triggers FraR dissociation from the DNA. These findings provide a first glimpse into the regulation of Amadori metabolism in a clinically significant bacterial pathogen.

Amadori rearrangement products are stable sugar-amino acid conjugates that are formed nonenzymatically during preparation, dehydration, and storage of foods. Because Amadori compounds such as fructose-lysine (F-Lys), an abundant constituent in processed foods, shape the animal gut microbiome, it is important to understand bacterial utilization of these fructosamines. In bacteria, F-Lys is first phosphorylated, either during or after uptake to the cytoplasm, to form 6-phosphofructose-lysine (6-P-F-Lys). FrlB, a deglycase, then converts 6-P-F-Lys to L-lysine and glucose-6-phosphate. Here, to elucidate the catalytic mechanism of this deglycase, we first obtained a 1.8-Å crystal structure of Salmonella FrlB (without substrate) and then used computational approaches to dock 6-P-F-Lys on this structure. We also took advantage of the structural similarity between FrlB and the sugar isomerase domain of Escherichia coli glucosamine-6-phosphate synthase (GlmS), a related enzyme for which a structure with substrate has been determined. An overlay of FrlB-6-P-F-Lys on GlmS-fructose-6-phosphate structures revealed parallels in their active-site arrangement and guided our selection of seven putative active-site residues in FrlB for site-directed mutagenesis. Activity assays with eight recombinant single-substitution mutants identified residues postulated to serve as the general acid and general base in the FrlB active site and indicated unexpectedly significant contributions from their proximal residues. By exploiting native mass spectrometry (MS) coupled to surface-induced dissociation, we distinguished mutations that impaired substrate binding versus cleavage. As demonstrated with FrlB, an integrated approach involving x-ray crystallography, in silico approaches, biochemical assays, and native MS can synergistically aid structure-function and mechanistic studies of enzymes.