Insights Into The Catalytic Mechanism of A Bacterial Deglycase Essential For Utilization of Fructose-Lysine.

Amadori rearrangement products are stable sugar-amino acid conjugates that are formed non-enzymatically 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 (Glc-6-P). 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-Fru-6-P 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. This article is protected by copyright. All rights reserved.