Variations in UDP-N-acetylglucosamine and UDP-N-acetylmuramyl-pentapeptide pools in Escherichia coli after inhibition of protein synthesis.

The pool levels of the nucleotide precursors of peptidoglycan were analyzed after inhibition of protein synthesis in various Escherichia coli strains. In all cases UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) cell pools increased upon treatment with chloramphenicol or tetracycline. Similar results were observed after the treatment of K-12 strains with valine. Since the intermediate nucleotide precursors did not accumulate after the arrest of protein synthesis and since a feedback mechanism was unlikely, the increases of the UDP-MurNAc-pentapeptide pool appeared as a consequence of that of the UDP-GlcNAc pool by the unrestricted functioning of the intermediate steps of the pathway. The highest increase (sixfold) of UDP-GlcNAc was observed with strain K-12 HfrH growing in minimal medium and treated with chloramphenicol. When a pair of isogenic Rel+ and Rel- strains were considered, both the UDP-GlcNAc and UDP-MurNAc-pentapeptide pools increased upon treatment with chloramphenicol or valine. However, the UDP-GlcNAc pool of the Rel+ strain was at a high natural level, which increased only moderately (20%) after the addition of valine. The increase of the UDP-GlcNAc pool after the various treatments could be due to an effect on some upstream step by an unknown mechanism. The possible correlations of the variations of the precursor pools with the rate of synthesis and extent of cross-linking of peptidoglycan were also considered.     

Mechanism of intrinsic resistance to vancomycin in Clostridium innocuum NCIB 10674

We have studied the basis for intrinsic resistance to low levels of vancomycin in Clostridium innocuum NCIB 10674 (MIC = 8 microg/ml). Analysis by high-pressure liquid chromatography (HPLC) and mass spectrometry of peptidoglycan nucleotide precursors pools revealed the presence of two types of UDP-MurNac-pentapeptide precursors constitutively produced, an UDP-MurNAc-pentapeptide with a serine at the C terminus which represented 93% of the pool and an UDP-MurNAc-pentapeptide with an alanine at the C terminus which represented the rest of the pool. C. innocuum cell wall muropeptides containing pentapeptide[Ser], either dialanine substituted on the epsilon amino group of lysine or not, were identified and represented about 10% of the monomers while only 1% of pentapeptide[D-Ala] monomers were found. The sequence of a 2,465-bp chromosomal fragment from C. innocuum was determined and revealed the presence of ddl(c. innocuum) and C. innocuum racemase genes putatively encoding homologues of D-Ala:D-X ligases and amino acid racemases, respectively. Analysis of the pool of precursors of Enterococcus faecalis JH2-2, containing cloned ddl(c. innocuum) and C. innocuum racemase genes showed in addition to the UDP-MurNAc-pentapeptide[D-Ala], the presence of an UDP-MurNAc-pentapeptide[D-Ser] precursor. However, the expression of low-level resistance to vancomycin was observed only when both genes were cloned in E. faecalis JH2-2 together with the vanXYc gene from Enterococcus gallinarum BM4174 which encodes a d,d-peptidase which eliminates preferentially the high affinity vancomycin UDP-MurNAc-pentapeptide [D-Ala] precursors produced by the host. We conclude that resistance to vancomycin in C. innocuum NCIB 10674 was related to the presence of the two chromosomal ddl(c. innocuum) and C. innocuum racemase genes allowing the synthesis of a peptidoglycan precursor terminating in serine with low affinity for vancomycin.     

Identification of the UDP-MurNAc-pentapeptide:L-alanine ligase for synthesis of branched peptidoglycan precursors in Enterococcus faecalis

Many species of gram-positive bacteria produce branched peptidoglycan precursors resulting from the transfer of various L-amino acids or glycine from amino acyl-tRNA to the epsilon-amino group of L-lysine. The UDP-MurNAc-pentapeptide:L-alanine ligase and alanyl-tRNA synthetase genes from Enterococcus faecalis were identified, cloned, and overexpressed in Escherichia coli. The purified enzymes were necessary and sufficient for tRNA-dependent addition of L-alanine to UDP-MurNAc-pentapeptide in vitro. The ligase belonged to the Fem family of proteins, which were initially identified genetically as factors essential for methicillin resistance in Staphylococcus aureus.     

Analysis of murein and murein precursors during antibiotic-induced lysis of Escherichia coli.

Lysis of Escherichia coli induced by either D-cycloserine, moenomycin, or penicillin G was monitored by studying murein metabolism. The levels of the soluble murein precursor UDP-N-acetylmuramyl-L-alanyl-D-glutamyl-m-diaminopimelyl-D-alanyl- D-alanine (UDP-MurNAc-pentapeptide) and the carrier-linked MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol as well as N-acetylglucosamine-beta-1,4-MurNAc-(pentapeptide)-pyrophosphoryl- undecaprenol varied in a specific way. In the presence of penicillin, which is known to interfere with the cross-linking of murein, the concentration of the lipid-linked precursors unexpectedly decreased before the onset of lysis, although the level of UDP-MurNAc-pentapeptide remained normal. In the case of moenomycin, which specifically blocks the formation of the murein polysaccharide strands, the lipid-linked precursors as well as UDP-MurNAc-pentapeptide accumulated as was expected. D-Cycloserine, which inhibits the biosynthesis of UDP-MurNAc-pentapeptide, consequently caused a decrease in all three precursors. The muropeptide composition of the murein showed general changes such as an increase in the unusual DL-cross bridge between two neighboring meso-diaminopimelic acid residues and, as a result of uncontrolled DL- and DD-carboxypeptidase activity, an increase in tripeptidyl and a decrease in tetrapeptidyl and pentapeptidyl moieties. The average length of the glycan strands decreased. When the glycan strands were fractionated according to length, a dramatic increase in the amount of single disaccharide units was observed not only in the presence of penicillin but also in the presence of moenomycin. This result is explained by the action of an exo-muramidase, such as the lytic transglycosylases present in E. coli. It is proposed that antibiotic-induced bacteriolysis is the result of a zipperlike splitting of the murein net by exo-muramidases locally restricted to the equatorial zone of the cell.     

Inhibition of uridine 5'-diphosphate-N-acetylmuramyl-L-alanine synthetase by beta-chloro-L-alanine in Escherichia coli

The synthesis of the nucleotide precursors for peptidoglycan is regulated by the relA gene in Escherichia coli. Thus, nucleotide precursors labeled with [3H]diaminopimelic acid accumulated in a relA strain but not in an isogenic relA+ strain during amino acid deprivation. Furthermore, nucleotide precursor synthesis was relaxed in the amino acid deprived relA+ strain by treatment with chloramphenicol. Uridine diphosphate-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) was the major component accumulated during the relaxed synthesis of nucleotide precursors in both relA+ and relA strains. The effect of beta-chloro-L-alanine (CLA) on the relaxed synthesis of nucleotide precursors for peptidoglycan was determined. At a low concentration (0.0625 mM) CLA inhibited the synthesis of UDP-MurNAc-pentapeptide and caused the accumulation of UDP-MurNAc-tripeptide. Thus, low concentrations of CLA probably inhibited alanine racemase, as reported previously. Higher concentrations of CLA also inhibited an earlier step in nucleotide precursor synthesis. This was shown to be due to the inhibition of UDP-MurNAc-L-alanine synthetase by CLA. CLA inhibited the activity of this enzyme in cell-free extracts as well as in intact cells.     

Purification and characterization of VanXY(C), a D,D-dipeptidase/D,D-carboxypeptidase in vancomycin-resistant Enterococcus gallinarum BM4174.

VanXY(C), a bifunctional enzyme from VanC-phenotype Enterococcus gallinarum BM4174 that catalyses D,D-peptidase and D,D-carboxypeptidase activities, was purified as the native protein, as a maltose-binding protein fusion and with an N-terminal tag containing six histidine residues. The kinetic parameters of His(6)-VanXY(C) were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for D-Ala-D-Ala, the K(m) was 3.6 mm and k(cat), 2.5 s(-1); for UDP-MurNAc-L-Ala-D-Glu-L-Lys-DAla-D-Ala (UDP-MurNAc-pentapeptide[Ala]), K(m) was 18.8 mm and k(cat) 6.2 s(-1); for D-Ala-D-Ser, K(m) was 15.5 mm and k(cat) 0.35 s(-1). His(6)-VanXYC was inactive against the peptidoglycan precursor UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ser (UDP-MurNAc-pentapeptide[Ser]). The rate of hydrolysis of the terminal D-Ala of UDP-MurNAc-pentapeptide[Ala] was inhibited 30% by 2 mm D-Ala-D-Ser or UDP-MurNAc-pentapeptide[Ser]. Therefore preferential hydrolysis of substrates terminating in D-Ala would occur during peptidoglycan synthesis in E. gallinarum BM4174, leaving precursors ending in D-Ser with a lower affinity for glycopeptides to be incorporated into peptidoglycan.Mutation of an aspartate residue (Asp59) of His-tagged VanXY(C) corresponding to Asp68 in VanX to Ser or Ala, resulted in a 50% increase and 73% decrease, respectively, of the specificity constant (k(cat)/K(m)) for D-Ala-D-Ala. This situation is in contrast to VanX in which mutation of Asp68-->Ala produced a greater than 200,000-fold decrease in the substrate specificity constant. This suggests that Asp59, unlike Asp68 in VanX, does not have a pivotal role in catalysis.     

One-step purification procedure for UDP-N-acetylmuramyl-peptide murein precursors from Bacillus cereus

A method is described for the rapid isolation of the activated murein precursors UDP-N-acetyl-muramyl-pentapeptide (UDP-MurNAc-pentapeptide) and UDP-MurNAc-tripeptide from Bacillus cereus. After accumulation of the precursors by inhibition of murein synthesis either in the presence of vancomycin (for the pentapeptide precursor) or D-cycloserine (for the tripeptide precursor) the cells were extracted with boiling water. Prior to high pressure liquid chromatography the material was freed from acid precipitable material. UDP-MurNAc-penta- and tripeptide were separated from other components by reversed-phase HPLC on Hypersil ODS using isocratic elution conditions with sodium phosphate buffer. The precursors were obtained with at least 98% purity and a yield of about 50 mumol from a 10-l culture of B. cereus.     

Presence of UDP-N-acetylmuramyl-hexapeptides and -heptapeptides in enterococci and staphylococci after treatment with ramoplanin, tunicamycin, or vancomycin.

Analyses of the peptidoglycan nucleotide precursor contents of enterococci and staphylococci treated with ramoplanin, tunicamycin, or vancomycin were carried out by high-pressure liquid chromatography coupled with mass spectrometry (MS). In all cases, a sharp increase in the UDP-N-actetylmuramoyl-pentapeptide or -pentadepsipeptide pool was observed. Concomitantly, new peptidoglycan nucleotide peptides of higher molecular masses with hexa- or heptapeptide moieties were identified: UDP-MurNAc-pentapeptide-Asp or pentadepsipeptide-Asp in enterococci and UDP-MurNAc-pentapeptide-Gly or -Ala and UDP-MurNAc-pentapeptide-Gly-Gly or -Ala-Gly in staphylococci. These new compounds are derivatives of normal UDP-MurNAc-pentapeptide or -pentadepsipeptide precursors with the extra amino acid(s) linked to the lysine epsilon-amino group as established by various analytical procedures (MS, MS-MS fragmentation, chemical analysis, and digestion with R39 D,D carboxypeptidase). Except for tunicamycin-treated cells, it was not possible to ascertain whether these unusual nucleotides were formed by direct addition of the amino acids to UDP-MurNAc-pentapeptide (or -pentadepsipeptide) or whether they arose by reverse reactions from lipid I intermediates to which the amino acids had been added.     

Determination of murein precursors during the cell cycle of Escherichia coli

A convenient and reliable method has been established that allows a quantitative determination of m-diamino[3H]pimelic acid-labelled murein precursors in 1 ml culture samples of Escherichia coli. Prior to separation by reversed-phase high-pressure liquid chromatography the lipid-linked intermediates were hydrolysed to release the muropeptides. The accuracy for the measurement of UDP-N-acetylmuramylpentapeptide (UDP-MurNAc-pentapeptide) was +/- 1.9% (SD), for undecaprenyl-P-P-MurNAc-pentapeptide (lipid I) +/- 10% (SD) and for undecaprenyl-P-P-(GlcNAc-beta 1----4)MurNAc-pentapeptide (lipid II) +/- 5% (SD). The ratio of UDP-MurNAc-pentapeptide:lipid I:lipid II was about 300:1:3 for E. coli MC4100. The relative cellular concentrations of all three precursor molecules were found not to vary throughout the cell cycle. It is concluded that elongation and division of the murein sacculus is not controlled by oscillations in the concentrations of these late murein precursors.     

Gene vanXYC encodes D,D -dipeptidase (VanX) and D,D-carboxypeptidase (VanY) activities in vancomycin-resistant Enterococcus gallinarum BM4174

VanX and VanY have strict D,D-dipeptidase and D,D-carboxypeptidase activity, respectively, that eliminates production of peptidoglycan precursors ending in D-alanyl-D-alanine (D-Ala-D-Ala) in glycopeptide-resistant enterococci in which the C-terminal D-Ala residue has been replaced by D-lactate. Enterococcus gallinarum BM4174 synthesizes peptidoglycan precursors ending in D-Ala-D-serine (D-Ala-D-Ser) essential for VanC-type vancomycin resistance. Insertional inactivation of the vanC-1 gene encoding the ligase that catalyses synthesis of D-Ala-D-Ser has a polar effect on both D, D-dipeptidase and D,D-carboxypeptidase activities. The open reading frame downstream from vanC-1 encoded a soluble protein designated VanXYC (Mr 22 318), which had both of these activities. It had 39% identity and 74% similarity to VanY in an overlap of 158 amino acids, and contained consensus sequences for binding zinc, stabilizing the binding of substrate and catalysing hydrolysis that are present in both VanX- and VanY-type enzymes. It had very low dipeptidase activity against D-Ala-D-Ser, unlike VanX, and no activity against UDP-MurNAc-pentapeptide[D-Ser], unlike VanY. The introduction of plasmid pAT708(vanC-1,XYC) or pAT717(vanXYC) into vancomycin-susceptible Enterococcus faecalis JH2-2 conferred low-level vancomycin resistance only when D-Ser was present in the growth medium. The peptidoglycan precursor profiles of E. faecalis JH2-2 and JH2-2(pAT708) and JH2-2(pAT717) indicated that the function of VanXYC was hydrolysis of D-Ala-D-Ala and removal of D-Ala from UDP-MurNAc-pentapeptide[D-Ala]. VanC-1 and VanXYC were essential, but not sufficient, for vancomycin resistance.     

Assay for identification of inhibitors for bacterial MraY translocase or MurG transferase

Bacterial peptidoglycan synthesis is a well-characterized system for targeting new antimicrobial drugs. Formation of the peptidoglycan precursors Lipid I and Lipid II is catalyzed by the gene products of mraY and murG, which are involved in the first and second steps of the lipid cycle reactions, respectively. Here we describe the development of an assay specific for identifying inhibitors of MraY or MurG, based on the detection of radiolabeled [(14)C]GlcNAc incorporated into Lipid II. Assay specificity is achieved with the biotin tagging of the Lipid I precursor UDP-MurNAc-pentapeptide. This allows for the separation and identification of lipid products produced by the enzymatic activity of the MraY and MurG proteins, and thus identification of specific inhibitors.     

Involvement of the relA gene product and feedback inhibition in the regulation of DUP-N-acetylmuramyl-peptide synthesis in Escherichia coli.

The regulation of uridine diphosphate-N-acetylmuramyl-peptide (UDP-MurNAc-peptide) synthesis was studied by labeling Escherichia coli strains auxotrophic for lysine and diaminopimelate with [3H]diaminopimelate for 15 min under various conditions. The amounts of [3H]diaminopimelate incorporated into UDP-MurNAc-tripeptide and -pentapeptide by a stringent (rel+) strain were the same in the presence or absence of lysine. Chloramphenicol-treated rel+ cells showed a 2.8-fold increase in labeled UDP-MurNAc-pentapeptide. An isogenic relaxed (relA) strain deprived of lysine showed a 2.7-fold increase in UDP-MurNAc-pentapeptide. Thus, UDP-MurNAc-pentapeptide synthesis is regulated by the relA gene. D-Cycloserine treatment of rel+ and relA strains caused a depletion of intracellular UDP-MurNAc-pentapeptide. Labeled UDP-MurNAc-tripeptide accumulated in D-cycloserine-treated cells of the rel+ and relA strains, suggesting that UDP-MurNAc-pentapeptide is a feedback inhibitor of UDP-MurNAc-peptide synthesis. In lysine-deprived cells, D-cycloserine treatment caused 41- and 71-fold accumulations of UDP-MurNAc-tripeptide in rel+ and relA strains, respectively. A 124-fold increase in UDP-MurNAc-tripeptide occurred in lysine-deprived rel+ cells treated with both chloramphenicol and D-cycloserine. These results indicate that both the relA gene product and feedback inhibition are involved in regulating UDP-MurNAc-peptide synthesis during amino acid deprivation.     

Structure-based site-directed mutagenesis of the UDP-MurNAc-pentapeptide-binding cavity of the FemX alanyl transferase from Weissella viridescens

Weissella viridescens FemX (FemX(Wv)) belongs to the Fem family of nonribosomal peptidyl transferases that use aminoacyl-tRNA as the amino acid donor to synthesize the peptide cross-bridge found in the peptidoglycan of many species of pathogenic gram-positive bacteria. We have recently solved the crystal structure of FemX(Wv) in complex with the peptidoglycan precursor UDP-MurNAc-pentapeptide and report here the site-directed mutagenesis of nine residues located in the binding cavity for this substrate. Two substitutions, Lys36Met and Arg211Met, depressed FemX(Wv) transferase activity below detectable levels without affecting protein folding. Analogues of UDP-MurNAc-pentapeptide lacking the phosphate groups or the C-terminal D-alanyl residues were not substrates of the enzyme. These results indicate that Lys36 and Arg211 participate in a complex hydrogen bond network that connects the C-terminal D-Ala residues to the phosphate groups of UDP-MurNAc-pentapeptide and constrains the substrate in a conformation that is essential for transferase activity.     

Occurrence and identification of UDP-N-acetylmuramyl-pentapeptide from the cyanobacterium Anabaena cylindrica

UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) is well known to be a key intermediate of bacterial peptidoglycan biosynthesis. We first detected the occurrence of UDP-MurNAc-pentapeptide in the cyanobacterium Anabaena cylindrica (NIES-19), and identified the structure of this pentapeptide by two-dimensional 1H-1H and 1H-13C NMR correlation experiments and mass spectra.     

The site of inhibition of bacterial cell wall peptidoglycan synthesis by azureomycin B, a new antibiotic

Azureomycin B (10 micrograms/ml), a new antibiotic from Pseudonocardia azurea nov. sp., caused the accumulation of lipid intermediate and inhibition of peptidoglycan synthesis in an invitro system using a particulate fraction from Bacillus megaterium KM with UDP-MurNAc-[3H]pentapeptide and cold UDP-GlcNac or cold UDP-MurNAc-pentapeptide and UDP-[3H]GlcNAc as substrates. At higher concentrations of azureomycin B (over 100 microgram/ml), lipid intermediate accumulation was also inhibited. When particulate fraction from Escherichia coli Y-10 and UDP-[14C[GlcNAc and cold UDP-MurNAc-pentapeptide were used, accumulation of lipid intermediate and inhibition of peptidoglycan synthesis were also observed. These results indicate that the primary target of azureomycin B is the transfer of the disaccharide peptide unit (GlcNAc-MurNAc-pentapeptide) from lipid-bound precursor to acceptor.     

Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3.

The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP 1b or PBP 3. With PBP 1b, tripeptide lipid II was used as efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP 1b activity accounts for at best to 2 to 3% of the in vivo synthesis. With PBP 3, no polymerization was observed with either substrate. Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP 1b could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.     

The murG gene of Escherichia coli codes for the UDP-N-acetylglucosamine: N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase involved in the membrane steps of peptidoglycan synthesis.

Physiological properties of the murG gene product of Escherichia coli were investigated. The inactivation of the murG gene rapidly inhibits peptidoglycan synthesis in exponentially growing cells. As a result, various alterations of cell shape are observed, and cell lysis finally occurs when the peptidoglycan content is 40% lower than that of normally growing cells. Analysis of the pools of peptidoglycan precursors reveals the concomitant accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) and, to a lesser extent, that of undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide (lipid intermediate I), indicating that inhibition of peptidoglycan synthesis occurs after formation of the cytoplasmic precursors. The relative depletion of the second lipid intermediate, undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)GlcNAc, shows that inactivation of the murG gene product does not prevent the formation of lipid intermediate I but inhibits the next reaction in which GlcNAc is transferred to lipid intermediate I. In vitro assays for phospho-MurNAc-pentapeptide translocase and N-acetylglucosaminyl transferase activities finally confirm the identification of the murG gene product as the transferase that catalyzes the conversion of lipid intermediate I to lipid intermediate II in the peptidoglycan synthesis pathway. Plasmids allowing for a high overproduction of the transferase and the determination of its N-terminal amino acid sequence were constructed. In cell fractionation experiments, the transferase is essentially associated with membranes when it is recovered.     

Biosynthesis of peptidoglycan in Gaffkya homari. The mode of action of penicillin G and mecillinam.

The effect of the beta-lactam antibiotics penicillin G and mecillinam on the incorporation of peptidoglycan into pre-formed cell wall peptidoglycan was studied with wall membrane enzyme preparations from Gaffkya homari. Using UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) as precursors the incorporation of peptidoglycan into the pre-existing cell wall of G. homari was inhibited to an extent of 50% (ID50 value) at a concentration of 0.25 mug of penicillin G/ml. With UDP-GlcNAc and UDP-MurNAc-tetrapeptide as precursors the ID50 value was about 2500-fold greater (630 mug/ml). The inhibition by penicillin G of the incorporation of peptidoglycan from UDP-MurNAc-[14C]Lys-pentapeptide could be overcome by addition of non-radioactive UDP-MurNAc-tetrapeptide to the incubation mixture. In the presence of 5 mug of penicillin G/ml the incorporation of peptidoglycan formed from the mixture of UDP-MurNAc-Ala-DGlu-Lys-D-[14C]Ala-D[14C]Ala and non-radioactive UDP-MurNAc-tetrapeptide proceeded virtually without release of D-[14C]alanine by transpeptidase activity. The enzyme preparation also exhibited DD-carboxypeptidase activity which was only slightly more sensitive to penicillin G and mecillinam than was the incorporation of peptidoglycan into the cell wall. Since the ID50 values for the beta-lactam antibiotics are similar to the concentrations required to inhibit the growth of G. homari to an extent of 50%, the DD-carboxypeptidase must be the killing site of both penicillin G and mecillinam.     

Crystal structures of Weissella viridescens FemX and its complex with UDP-MurNAc-pentapeptide: insights into FemABX family substrates recognition

Members of the FemABX protein family are novel therapeutic targets, as they are involved in the synthesis of the bacterial cell wall. They catalyze the addition of amino acid(s) on the peptidoglycan precursor using aminoacylated tRNA as a substrate. We report here the high-resolution structure of Weissella viridescens L-alanine transferase FemX and its complex with the UDP-MurNAc-pentapeptide. This is the first structure example of a FemABX family member that does not possess a coiled-coil domain. FemX consists of two structurally equivalent domains, separated by a cleft containing the binding site of the UDP-MurNAc-pentapeptide and a long channel that traverses one of the two domains. Our structural studies bring new insights into the evolution of the FemABX and the related GNAT superfamilies, shed light on the recognition site of the aminoacylated tRNA in Fem proteins, and allowed manual docking of the acceptor end of the alanyl-tRNAAla.