Structural Basis for EarP-Mediated Arginine Glycosylation of Translation Elongation Factor EF-P

Abstract

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-β-l-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design.

Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P

“…Asp16 of P. aeruginosa EarP corresponds to Asp20 of N. meningitidis EarP, which has been reported as the general base (30). Moreover, replacement of Glu273 of P. putida EarP, corresponding to Glu272 of P. aeruginosa EarP, with a glutamine resulted in undetectable EF-P rhamnosylation in vivo (29). To further analyze the roles of these conserved key residues, we performed site-directed mutagenesis combined with an in vitro assay for EF-P rhamnosylation, detected by mass spectrometry.…”

“…During the preparation of our manuscript, two independent works on the structure of EarP appeared. R. Krafczyk et al were the first to report the crystal structure of EarP from Pseudomonas putida bound to TDP-Rha (29). These researchers identified that EarP contains two opposing Rossmann fold domains, which classify EarP in glycosyltransferase superfamily B (GT-B).…”

Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3

“…Mutation into an alanine residue, similarly to the mutation of the equivalent residue in NleB1 (E253A), impairs the arginine GlcNAcylation ability of SseK3 and NleB1 without, for the latter, abrogating binding to the target protein ( 22 ). To date, the only available structure of an arginine-glycosylation enzyme is that of the Pseudomonas aeruginosa EarP that catalyzes the transfer of a rhamnose molecule onto the bacterial translation elongation factor EF-P ( 16 ). EarP is an inverting glycosyltransferase belonging to the GT-B family and structurally unrelated to SseK3.…”

“…Interestingly, EarP has three negatively charged residues in the active site (Asp-13, Asp-17, and Glu-273), in close proximity to the sugar, that have been shown to be important in the catalytic activity and play a crucial role in the stabilization of the positive charge of the acceptor guanidino group. Mutation of those residues to alanine, as for the E258A mutation in SseK3, severely impairs EarP enzymatic activity without disrupting substrate binding ( 16 ).…”

“…Although, to date, there are a number of structures describing O -glycosyltransferases and the O -GlcNAcylation catalytic event, the only known structure of a bacterial arginine-specific glycosyltransferase is that of the B pattern-type inverting glycosyltransferase EarP, which modifies translation elongation factor P (EF-P) by arginine rhamnosylation ( 15 , 16 ). Another study reported glucosylation of arginine by the sweet corn protein amylogenin, which mediates self-glucosylation in vitro ( 17 ), but there have been no follow-up reports.…”