Figure 1: EF-P - Glyco­sylation and mode of action A EF-P binds to the diproline-locked ribosome. The interaction of α-rhamnosyl arginine with the CCA end of the P-site tRNA facilitates the formation of a new peptide bond.
B Posttranslational activation of EF-P by the glycosyltransferase EarP of P. putida with dTDP-β-L-rhamnose (TDP-Rha). Depicted is the catalytic pocket with its crucial aspartates D13 and D17, priming arginine R32 for the nucleophilic attack onto the anomeric carbon of TDP-Rha to mediate the formation of α-rhamnosyl arginine.

PD Dr. Jürgen Lassak
Area B Synthetic Proteins

What a bacterial translation elongation factor teaches us about glycosylation

Glycosylations are one of the most important and probably the most frequent posttranslational protein modifications in biological systems. Nevertheless, for a long time it was believed that they were limited to eukaryotes. However, bacteria also contain a large number of glycoproteins that fulfil important functions, for example in protein bio­synthesis. This process comes to an unwanted stop when two or more consecutive prolines have to be translated. The evolutionary solution to this problem is a specialized protein that is recruited to the arrested ribosome and restarts the elongation of the amino acid chain (Fig. 1A). Especially important for the activity of this universal elongation factor, in bacteria EF-P, are posttranslational modifications. They play a decisive role in the stabilization and orientation of peptidyl-tRNA in order to form a new peptide bond. In pseudomonads and thus also in the pathogen of bacterial pneumonia P. aeruginosa, a conserved arginine is α-rhamnosylated by the novel glycosyltransferase EarP. Hereby the nucleotide precursor dTDP-β-L-rhamnose serves as donor substrate.

However, our discovery is not only interesting with regard to the functionalization of EF-P. On the one hand, rhamnosylation plays a crucial role for bacterial pathogenicity and thus makes EarP an interesting target structure for the development of new antimicrobial agents. On the other hand, it creates a precedent in (bacterial) glycobiology. The literature on nitrogen-linked protein glycosylations is almost exclusively limited to asparagine as acceptor amino acid and EF P is still the only known arginine glycosylated protein in bacteria. Together with a research team led by Janosch Hennig, we were recently able to solve the crystal structure of EarP and thus understand for the first time how glycosyl transfer to arginine is possible. Surprisingly, the postulated catalysis mechanism is similar to that of known asparagine glycosyltransferases (Fig. 1B). It therefore stays an open question to what extent arginine in fact remains the acceptor exception from the rule of N-linked glycosylations. Equally exciting is the examination of EF-P rhamnosylation from the sugar perspective. Just like arginine as acceptor amino acid, activated rhamnose as donor substrate appears to be rather unusual, despite its ubiquitous occurrence.

Together with Prof. Dr. Anja Hoffmann-Röder and her colleagues Daniel Gast and Swetlana Wunder, we are currently investigating the natural distribution of rhamnosylations and actually have first indications of new glycoproteins, for example in mycobacteria. In contrast to most other prokaryotes, here the dTDP-β-L-rhamnose biosynthesis pathway is essential. So far, it has been assumed that this is due to a special structure in the cell wall, the mycolic acids, which are linked to the peptidoglycan by rhamnose. Alternatively, mycobacteria may also contain an essential protein whose activity is directly linked to posttranslational glycosylation, similar to EF-P in pseudomonads. Should this be confirmed, this would perhaps be a new target for the fight against tuberculosis. Our finding also underlines once again the importance of research on bacterial glycosylations.