New pathways for the metabolism of fructosamines and other glycation products in bacteria
(2005)
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- Authors
- Supervisors
- Van Schaftingen, Emile
- Abstract
- Glucose reacts spontaneously with amines of amino acids and proteins to form Schiff bases, which slowly undergo an Amadori rearrangement that converts them to fructosamines. This process, known as glycation, is observed with all types of reducing sugars. When glycation implicates fructose, the main rearrangement product, known as Heyns product, has a glucosamine-like structure. Before this thesis work was started, fructosamines were known to be metabolized in some bacteria and fungi by fructosyl-amino acid oxidases that most often convert fructosamines to glucosone and a free amine with production of H2O2. In mammals, fructosamine 3-kinase is known to catalyze the phosphorylation of fructosamines to fructosamine 3-phosphates, which spontaneously degrade to 3-deoxyglucosone, inorganic phosphate and a free amine. The seminal observation of the present thesis was the observation that Escherichia coli extracts catalyze the ATP-dependent phosphorylation of fructoselysine. This prompted us to study fructoselysine metabolism in this bacterium. We could show that this Amadori product supports the growth of E. coli, and that fructoselysine is phosphorylated in cell extracts to a compound that is converted to glucose 6-phosphate. The operon encoding the implicated enzymes was identified and given the designation frl. FrlA encodes a putative transporter. FrlD is a kinase of the PfkB family that was characterized and shown to phosphorylate fructoselysine to fructoselysine 6-phosphate. The latter is converted to glucose 6-phosphate and free lysine by fructoselysine-6-phosphate deglycase, the product of the frlB gene, which is distantly related to the isomerase domain of glucosamine-6-phosphate synthase. FrlC is homologous to tagatose 3-epimerase, which led us to hypothesize that it catalyzes the isomerization of fructoselysine and its C3 epimer, psicoselysine. This hypothesis was confirmed by overexpressing FrlC and characterizing its enzymatic activity. Furthermore, wild-type E. coli was found to grow on psicoselysine, but this ability (as well as that of growing on fructoselysine) was lost in two mutants of the frl operon, confirming the involvement of this operon in the metabolism of both lysine derivatives. A study of the Bacillus subtilis homologues of FrlD and FrlB, known as YurL and YurP, respectively, allowed us to demonstrate that they catalyze similar reactions as their E. coli counterpart, but that their specificity is totally different. Instead of acting on fructoselysine (or its 6-phospho-derivative), they preferentially act on fructosamine derivatives of hydrophobic a-amino acids. Finally, we noted that several bacterial operons that contain a putative fructosamine-6-phosphate deglycase also encode a second, closer homologue of the isomerase domain of glucosamine-6-phosphate synthase. Overexpression and characterization of these two proteins (encoded by an Enterococcus faecium operon) allowed us to show that the first one catalyzes the cleavage of fructoselysine 6-phosphate to glucose 6-phosphate and lysine and the second one, the cleavage of glucoselysine 6-phosphate to fructose 6-phosphate and lysine. Operons encoding this pair of enzymes also comprise a phosphotransferase system, which is presumably responsible for the transport and phosphorylation of both fructoselysine and glucoselysine. This new type of operon appears therefore to be responsible for the metabolism of both glycation and fructation products. In conclusion, our work has led to the identification of new enzymes involved in the metabolism of glycation products. These enzymes appear to be more common than fructosyl-amino acid oxidases in the bacterial world. Furthermore, they provide interesting tools for the assay and production of fructosamines and related compounds. ...
- Affiliations
Citations
Wiame, E. (2005). New pathways for the metabolism of fructosamines and other glycation products in bacteria. https://hdl.handle.net/2078.5/110697