Active Site Mapping of MraY, a Member of the Polyprenyl-phosphate N-Acetylhexosamine 1-Phosphate Transferase Superfamily, Catalyzing the First Membrane Step of Peptidoglycan Biosynthesis

Abstract

The MraY transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. No 3D structure has been reported for any member of this superfamily, and to date MraY is the only protein that has been successfully purified to homogeneity. Nineteen polar residues located in the five cytoplasmic segments of MraY appeared as invariants in the sequences of MraY orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the Bacillus subtilis MraY as a model. Fourteen residues were shown to be essential for MraY activity by an in vivo functional complementation assay using a constructed conditional mraY mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of the nucleotidic and lipidic substrates, but the k cat was dramatically reduced in almost all cases. The important residues for activity therefore appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the D98 residue that is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process.

Preparative Scale Cell-free Production and Quality Optimization of MraY Homologues in Different Expression Modes

“…Lipid I is assembled on the inner surface of the cytoplasmic membrane as the first lipid-linked intermediate of peptidoglycan synthesis by transfer of the phospho-MurNAc-pentapeptide moiety of UDP-MurNAc-pentapeptide to the membrane anchor C 55 -P (25-27). This first membrane-bound step is catalyzed by the integral translocase MraY (3,28). MraY is essential for bacterial viability as well as a ubiquitous presence in the eubacterial kingdom.…”

A haploid genetic screen identifies the major facilitator domain containing 2A (MFSD2A) transporter as a key mediator in the response to tunicamycin

“…Its catalytic center has been proposed to act on the cytoplasmic side of the ER (49,50). Cytoplasmic segments are also important for the catalytic activity of MraY, the bacterial homolog of DPAGT1/GPT (51). Thus, it is likely that, for TM to inhibit N-glycosylation, it does not need to permeate into the ER lumen.…”

5′-Methylene-triazole-substituted-aminoribosyl uridines as MraY inhibitors: synthesis, biological evaluation and molecular modeling

“…Both of them revealed a good interaction between the amino-ribosyl part of inhibitor 25a and the Mg 2+ cation (green ball, Fig. 10) as well as a varied and complex network of low electrostatic interactions with residues located in the active site and known to be important for substrate recognition, 13 such as Asp265 or Lys121. A major difference between both modes of interaction is the positioning of The effect of introducing a hydrophobic moiety on an aminoribosyl scaffold has already been reported.…”

“…Nevertheless, the challenge has recently been addressed and the 3D crystal structure of MraY from Aquifex aeolicus (MraY AA ) has been determined, 11 confirming key structural features that had been previously identified. 12,13 This enzyme, which catalyzes the first membrane-associated step of peptidoglycan biosynthesis, has been demonstrated to be essential 14 and is ubiquitous in the bacterial kingdom.…”