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IntroductionMraY, called also phospho-N-acetylmuramoyl-pentapeptide-transferase or UDP-MurNAc-pentapeptide phosphotransferase, with EC number 2.7.8.13, is an integral membrane enzyme involved in peptidoglycan biosynthesis [1] [2].MraY is encoded by the mraY gene and belongs to a subfamily of the polyprenyl-phosphate N-acetyl hexosamine 1-phosphate transferase (PNPT) superfamily [3]. MRAY is a promising candidate for the development of new antibiotics. In fact, it is the target of five classes of natural nucleoside inhibitors with potent antibacterial activity: the liposidomycins/caprazamycins, capuramycins, mureidomycins, muraymycins, and tunicamycins [2] [4].The structure presented in this page correspond to the MraY protein from Aquifex aeolicus (strain VF5), expressed in Escherichia coli, in complex with carbacaprazamycin .Carbacaprazamycin is a chemically stable analog of caprazamycin nucleoside inhibitors [5]. Drug resistant bacteria are the cause of death of millions of people worldwide. In the USA alone, hospital infections associated with antibiotic-resistant pathogens cause 99 000 deaths per year[6]. The development of new antibiotics with new mechanism of action is urgent. Structural analysis of the binding of carbacaprazamycin to MraY provides a better understanding of the chemical logic of MraY inhibition, which can help in the development of novel approaches for the design of antibiotics targeting MraY.
Functionbacteria [7]. The cell wall provides bacteria a structural support and protection. In particular, it allows bacteria to maintain their cell shape at different osmotic pressures [8]. Peptidoglycan is a cross-linked polymer of carbohydrates an amino acids and due to its biological relevance in Bacteria, it has been a major target for antibiotics [3] [7]. Peptidoglycan biosynthesis involves three main stages. MraY is responsible for the second stage. First, the peptidoglycan precursor UDP-Nacetylmuramoyl (MurNAc)–pentapeptide is synthesized in the cytosol. Second, this hydrophilic precursor is attached to a lipid carrier, and the complex lipid carrier-precursor is transported, through the membrane, to the periplasm. Third, the peptidoglycan precursors are polymerized to form the cell wall. MraY catalyzes the transfer of phospho-MurNAc-pentapeptide from hydrophilic substrate UDP-MurNAc-pentapeptide to the lipid carier (C55-P) in the presence of a Mg2+ cofactor. The product is the undecaprenyl-pyrophosphoryl-MurNAcpentapeptide, also known as lipid I [3] [9] [10]. StructureMraY is a dimeric membrane-bound enzyme, for which the N and the C termini are located on the periplasmic side. This protein is made of four extracellular loops, five cytoplasmic loops (named A,B … and E) and ten transmembrane helices named TM1 to TM10. Though, TM9 is cleaved by a glycin residue into two helical segments named TM9a and TM9b. Furthermore, there is an additional helix between TM9b and TM10, which is only 11 residues long and is called TM9c. This helix contains a HHH motif (H324,325 and 326) which plays a role in the enzyme’s substrat selectivity and is a part of the catalytic site [1]. For example; they interact with tunicamycin and MD2. This small loop binds two Ni2+ ions, one on the two first histidines and the other one on the last one. Another part of this site is the residues corresponding to Asp117 and Asp118 which are involved in Mg2+coordination [4].TM5–TM10 and loops C and D also play a role in the catalytic site and contain many polar and charged amino acids residues [1]. Also, some polar and charged amino acids on TM9b and loop E are pointing toward the active site, making it even more hydrophilic [4]. Carbacaprazamycin is made of uridine, 5-aminoribosyl, diazepanone, and aliphatic tail moieties. The diazepanone ring system makes relatively few interactions with the protein. Carbacapzazamycin contains an uridine binding pocket which is formed by amino acid residues in Loop C ( G194, L195, and D196). This pocket is capped off by a π–π stacking interaction with F262 in Loop D. K70 forms an additional hydrogen bond with the uracil moiety. Next to the uridine binding site, there is a second binding pocket lined with amino acid residues T75, N190, D193, and G264, called the uridine-adjacent pocket. The 5-aminoribose moiety of carbacaprazamycin forms an extensive hydrogen bond network in the uridine-adjacent pocket [9].
3D related structures
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