Crystal Structure of MraY, an Essential Membrane Enzyme for Bacterial Cell Wall Synthesis

Abstract

MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg2+ within the active site, and provide a structural basis of catalysis for this class of enzyme.

Crystal structure of lipid phosphatase Escherichia coli phosphatidylglycerophosphate phosphatase B

“…Recently, the crystal structure of phospho-N-acetylmuramic acid-pentapeptide translocase (MraY) from Aquifex aeolicus, which catalyzes the transfer of substrates to C 55 -P, was reported (11). For this process to be sustainable, the transport intermediate, undecaprenyl pyrophosphate, needs to be dephosphorylated on the periplasmic side by a phosphatase, for example, phosphatidyl-glycero-phosphatase B (PgpB, EC 3.1.3.27) (12), which may also be involved in the biogenesis of phosphatidylglycerol from phosphatidylglycerol phosphate (13).…”

Inhibition of phospho-MurNAc-pentapeptide translocase (MraY) by nucleoside natural product antibiotics, bacteriophage ϕX174 lysis protein E, and cationic antibacterial peptides

“…However, interestingly, the RWoct dipeptide derivative has antimicrobial activity against E. coli and P. aeruginosa [48]. In the crystal structure of A. aeolicus MraY [8], close to Phe-288 there are three other Phe residues forming a hydrophobic pocket, that may assist in the protein-protein interaction site, that are conserved in other Gram-negative MraY sequences. We note in Table 2 that many of the antibacterial peptides containing Arg-Trp sequences show activity against P. aeruginosa as well as E. coli.…”

“…aeolicus MraY [8] opens up possibilities for structure-based drug design against MraY and related enzymes. Although protein biochemistry for this group of integral membrane proteins remains very challenging, Bernard and co-workers have shown that recombinant MraY enzymes can be produced in good yield via cell-free protein expression [64], and can be reconstituted in nanodisc synthetic bilayers [68], offering an alternative to conventional protein expression technology.…”

“…Replacement of each of these residues by Asn gives inactive enzyme [6,7], indicating their essential function in catalysis, and it has been proposed that two Asp residues may be involved in binding of the Mg 2+ cofactor, and the other may be a catalytic nucleophile [6]. The crystal structure for Aquifex aeolicus MraY was determined in 2013 by Chung et al to 3.3 Å resolution [8]. The enzyme was crystallised as a dimer, with an inter-dimer interface involving helices 10 and 7 (see Figure 2A).…”

“…At the active site of the enzyme, the three catalytic aspartic acid residues are positioned in the centre of the active site, close to the Mg 2+ cofactor, which is ligated to Asp-265 (see Figure 2B). Polar residues Glu-300 and Gln-307 point into the active site from bent helix 9, and two histidine residues His-324 and His-325 on a large cytoplasmic loop also point into the active site [8].…”