Understanding the metabolism of Nε-carboxymethyllysine in Escherichia coli

• Applicants: Professor Dr. Michael Hellwig; Privatdozent Dr. Jürgen Lassak
• Subject Area: Food Chemistry ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 504633492

During food processing, reactions with reducing sugars (Maillard reaction) lead to covalent modification of proteins. While several individual glycated amino acids have already been analyzed in food both qualitatively and quantitatively, knowledge about the nutritional consequences of the consumption of the respective products, especially advanced glycation end products (AGEs), has remained fragmentary. The lack of knowledge also comprises bacterial degradability of the mostly unabsorbable modified amino acids. First studies indicate a decarboxylation of the AGE Nε-carboxymethyllysine (CML) in Escherichia coli.We have now identified the decarboxylases SpeC and SpeF as the first CML-degrading enzymes in E. coli. The significant differences between CML and the enzymes’ cognate substrate ornithine indicate for a broad range of substrates. We hypothesize that Nε-modified lysine derivatives are substrates of both decarboxylases as long as the nitrogen atom in the side-chain can be protonated. Therefore, our first aim is to investigate which additional modified amino acids (e.g. Nε-methyllysine, Nε-acetyllysine) are decarboxylated by SpeC and SpeF. The substrate conversion is measured by chromatographic methods to derive kinetic parameters.The reported occurrence of minor metabolites of CML suggests further metabolism of the intermediate biogenic amines. Similar to decarboxylation, these redox reactions and transaminations are most likely catalyzed by promiscuous enzymes. Therefore, we will focus on known transaminases and aldehyde dehydrogenases and test for their contribution to the metabolism of modified amino acids. For this purpose, potential metabolites will be synthesized in order to use them for chemical analyses and in studies on bacterial metabolism. As CML can be used as a nitrogen source in E. coli, we will identify CML-degrading enzymes by testing deletion mutants from the Keio collection. HPLC-MS/MS and GC-MS will be used for metabolite analysis.Our data also indicate that metabolism initiated with the SpeC/SpeF-mediated decarboxylation reaction cannot account for the total degradation of CML observed in E. coli. For this reason, supernatants from E. coli cultures, grown with and without CML as medium supplement, are subjected to untargeted metabolomics by HPLC-ESI-TOF-MS/MS. We hypothesize that also low-molecular weight compounds such as acetate and glyoxylate are formed from CML along with other metabolites. In order to reveal the origin of acetate, glyoxylate and other low-molecular weight compounds, we rely on various CML isotopologues that will be synthesized in the project.In summary, we will gain new insights into the metabolism of CML in E. coli which in turn will allow for conclusions about its degradation in bacteria in general. From a food-chemical and nutritional-physiological perspective, the transfer to the intestinal metabolism will enrich current discussions about the health relevance of modified amino acids.

• DFG Programme: Research Grants