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Bacillus subtilis PdaC (BsPdaC) is a membrane-bound, multidomain peptidoglycan N-deacetylase acting on N-acetylmuramic acid (MurNAc) residues and conferring lysozyme resistance to modified cell wall peptidoglycans. BsPdaC contains a C-terminal family 4 carbohydrate esterase (CE4) catalytic domain, but unlike other MurNAc deacetylases, BsPdaC also has GlcNAc deacetylase activity on chitooligosaccharides (COSs), characteristic of chitin deacetylases. To uncover the molecular basis of this dual activity, here we determined the X-ray structure of the BsPdaC CE4 domain at 1.54 Å resolution and analyzed its mode of action on COS substrates. We found that the minimal substrate is GlcNAc3 and that activity increases with the degree of glycan polymerization. COS deacetylation kinetics revealed that BsPdaC operates by a multiple-chain mechanism starting at the internal GlcNAc units and leading to deacetylation of all but the reducing-end GlcNAc residues. Interestingly, BsPdaC shares higher sequence similarity with the peptidoglycan GlcNAc deacetylase SpPgdaA than with other MurNAc deacetylases. Therefore, we used ligand-docking simulations to analyze the dual GlcNAc- and MurNAc-binding specificities of BsPdaC and compared them with those of SpPgdA and BsPdaA, representing peptidoglycan deacetylases highly specific for GlcNAc or MurNAc residues, respectively. BsPdaC retains the conserved Asp-His-His metal-binding triad characteristic of CE4 enzymes acting on GlcNAc residues, differing from MurNAc deacetylases that lack the metal-coordinating Asp residue. BsPdaC contains short loops similar to those in SpPgdA, resulting in an open binding cleft that can accommodate polymeric substrates. We propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases.

Endo-β-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the β-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-β-N-acetylmuramidases, which catalyze an exo-lytic cleavage of β-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-β-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl β-MurNAc and the peptidoglycan-derived disaccharide MurNAc-β-1,4-GlcNAc. Together with the exo-β-N-acetylglucosaminidase NagZ and the exo-muramoyl-l-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171; www.cazy.org/GH171.html). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria.

The N-acetylmuramic acid α-1-phosphate (MurNAc-α1-P) uridylyltransferase MurU catalyzes the synthesis of uridine diphosphate (UDP)-MurNAc, a crucial precursor of the bacterial peptidoglycan cell wall. MurU is part of a recently identified cell wall recycling pathway in Gram-negative bacteria that bypasses the general de novo biosynthesis of UDP-MurNAc and contributes to high intrinsic resistance to the antibiotic fosfomycin, which targets UDP-MurNAc de novo biosynthesis. To provide insights into substrate binding and specificity, we solved crystal structures of MurU of Pseudomonas putida in native and ligand-bound states at high resolution. With the help of these structures, critical enzyme-substrate interactions were identified that enable tight binding of MurNAc-α1-P to the active site of MurU. The MurU structures define a "minimal domain" required for general nucleotidyltransferase activity. They furthermore provide a structural basis for the chemical design of inhibitors of MurU that could serve as novel drugs in combination therapy against multidrug-resistant Gram-negative pathogens.

Tannerella forsythia is a Gram-negative periodontal pathogen lacking the ability to undergo de novo synthesis of amino sugars N -acetylmuramic acid (MurNAc) and N -acetylglucosamine (GlcNAc) that form the disaccharide repeating unit of the peptidoglycan backbone. T. forsythia relies on the uptake of these sugars from the environment, which is so far unexplored. Here, we identified a novel transporter system of T. forsythia involved in the uptake of MurNAc across the inner membrane and characterized a homolog of the Escherichia coli MurQ etherase involved in the conversion of MurNAc-6-phosphate (MurNAc-6-P) to GlcNAc-6-P. The genes encoding these components were identified on a three-gene cluster spanning Tanf_08375 to Tanf_08385 located downstream from a putative peptidoglycan recycling locus. We show that the three genes, Tanf_08375, Tanf_08380, and Tanf_08385, encoding a MurNAc transporter, a putative sugar kinase, and a MurQ etherase, respectively, are transcriptionally linked. Complementation of the Tanf_08375 and Tanf_08380 genes together in trans , but not individually, rescued the inability of an E. coli mutant deficient in the phosphotransferase (PTS) system-dependent MurNAc transporter MurP as well as that of a double mutant deficient in MurP and components of the PTS system to grow on MurNAc. In addition, complementation with this two-gene construct in E. coli caused depletion of MurNAc in the medium, further confirming this observation. Our results show that the products of Tanf_08375 and Tanf_08380 constitute a novel non-PTS MurNAc transporter system that seems to be widespread among bacteria of the Bacteroidetes phylum. To the best of our knowledge, this is the first identification of a PTS-independent MurNAc transporter in bacteria. IMPORTANCE In this study, we report the identification of a novel transporter for peptidoglycan amino sugar N -acetylmuramic acid (MurNAc) in the periodontal pathogen T. forsythia . It has been known since the late 1980s that T. forsythia is a MurNAc auxotroph relying on environmental sources for this essential sugar. Most sugar transporters, and the MurNAc transporter MurP in particular, require a PTS phosphorelay to drive the uptake and concurrent phosphorylation of the sugar through the inner membrane in Gram-negative bacteria. Our study uncovered a novel type of PTS-independent MurNAc transporter, and although so far, it seems to be unique to T. forsythia , it may be present in a range of bacteria both of the oral cavity and gut, especially of the phylum Bacteroidetes .

International audience; Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg2+ ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.

Inactivation of sll0861 in Synechocystis sp. strain PCC 6803 or the homologous gene alr2432 in Anabaena sp. strain PCC 7120 had no effect on the growth of these organisms at a light intensity of 30 μmol photons m −2 s −1 but reduced their growth at a light intensity of 5 or 10 μmol photons m −2 s −1 . In Anabaena , inactivation of the gene also significantly reduced the rate of heterocyst differentiation under low-light conditions. The predicted products of sll0861 and alr2432 and homologs of these genes showed similarity to N -acetylmuramic acid 6-phosphate etherase (MurQ), an enzyme involved in peptidoglycan recycling, in Escherichia coli. E. coli murQ and the cyanobacterial homologs could functionally substitute for each other. We hypothesize that murQ in cyanobacteria promotes low-light adaptation through reutilization of peptidoglycan degradation products.

The MurNAc etherase MurQ of Escherichia coli is essential for the catabolism of the bacterial cell wall sugar N -acetylmuramic acid (MurNAc) obtained either from the environment or from the endogenous cell wall (i.e., recycling). High-level expression of murQ is required for growth on MurNAc as the sole source of carbon and energy, whereas constitutive low-level expression of murQ is sufficient for the recycling of peptidoglycan fragments continuously released from the cell wall during growth of the bacteria. Here we characterize for the first time the expression of murQ and its regulation by MurR, a member of the poorly characterized RpiR/AlsR family of transcriptional regulators. Deleting murR abolished the extensive lag phase observed for E. coli grown on MurNAc and enhanced murQ transcription some 20-fold. MurR forms a stable multimer (most likely a tetramer) and binds to two adjacent inverted repeats within an operator region. In this way MurR represses transcription from the murQ promoter and also interferes with its own transcription. MurNAc-6-phosphate, the substrate of MurQ, was identified as a specific inducer that weakens binding of MurR to the operator. Moreover, murQ transcription depends on the activation by cyclic AMP (cAMP)-catabolite activator protein (CAP) bound to a class I site upstream of the murQ promoter. murR and murQ are divergently orientated and expressed from nonoverlapping face-to-face (convergent) promoters, yielding transcripts that are complementary at their 5′ ends. As a consequence of this unusual promoter arrangement, cAMP-CAP also affects murR transcription, presumably by acting as a roadblock for RNA polymerase.

Geobacillus tepidamans GS5-97(T) is a novel Gram-positive, moderately thermophilic bacterial species that is covered by a glycosylated surface layer (S-layer) protein. The isolated and purified S-layer glycoprotein SgtA was ultrastructurally and chemically investigated and showed several novel properties. By SDS-PAGE, SgtA was separated into four distinct bands in an apparent molecular mass range of 106-166 kDa. The three high molecular mass bands gave a positive periodic acid-Schiff staining reaction, whereas the 106-kDa band was nonglycosylated. Glycosylation of SgtA was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and electrospray ionization quadrupole time-of-fight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [--2)-alpha-L-Rhap-(1--3)-alpha-D-Fucp-(1--](n=approximately 20), with D-fucopyranose having never been identified before as a constituent of S-layer glycans. The rhamnose residue at the nonreducing end of the terminal repeating unit of the glycan chain was di-substituted. For the first time, (R)-N-acetylmuramic acid, the key component of prokaryotic peptidoglycan, was found in an alpha-linkage to carbon 3 of the terminal rhamnose residue, serving as capping motif of an S-layer glycan. In addition, that rhamnose was substituted at position 2 with a beta-N-acetylglucosamine residue. The S-layer glycan chains were bound via the trisaccharide core --2)-alpha-L-Rhap-(1--3)-alpha-L-Rhap-(1--3)-alpha-L-Rhap-(1--to carbon 3 of beta-D-galactose, which was attached in O-glycosidic linkage to serine and threonine residues of SgtA of G. tepidamans GS5-97(T).

A polysaccharide deacetylase homologue, PdaA, was determined to act as anN-acetylmuramic acid deacetylase in vitro. Histidine-tagged truncated PdaA (with the putative signal sequence removed) was overexpressed inEscherichia colicells and purified. Measurement of deacetylase activity showed that PdaA could deacetylate peptidoglycan treated withN-acetylmuramoyl-l-alanine amidase CwlH but could not deacetylate peptidoglycan treated with or withoutdl-endopeptidase LytF (CwlE). Reverse-phase high-performance liquid chromatography and mass spectrometry (MS) and MS-MS analyses indicated that PdaA could deacetylate theN-acetylmuramic acid residues of purified glycan strands derived fromBacillus subtilispeptidoglycan.

We report here that wild-type Escherichia coli grows on N -acetylmuramic acid (MurNAc) as the sole source of carbon and energy. Analysis of mutants defective in N -acetylglucosamine (GlcNAc) catabolism revealed that the catabolic pathway for MurNAc merges into the GlcNAc pathway on the level of GlcNAc 6-phosphate. Furthermore, analysis of mutants defective in components of the phosphotransferase system (PTS) revealed that a PTS is essential for growth on MurNAc. However, neither the glucose-, mannose/glucosamine-, nor GlcNAc-specific PTS (PtsG, ManXYZ, and NagE, respectively) was found to be necessary. Instead, we identified a gene at 55 min on the E. coli chromosome that is responsible for MurNAc uptake and growth. It encodes a single polypeptide consisting of the EIIB and C domains of a so-far-uncharacterized PTS that was named murP . MurP lacks an EIIA domain and was found to require the activity of the crr -encoded enzyme IIA-glucose (EIIA Glc ), a component of the major glucose transport system for growth on MurNAc. murP deletion mutants were unable to grow on MurNAc as the sole source of carbon; however, growth was rescued by providing murP in trans expressed from an isopropylthiogalactopyranoside-inducible plasmid. A functional His 6 fusion of MurP was constructed, isolated from membranes, and identified as a polypeptide with an apparent molecular mass of 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Close homologs of MurP were identified in the genome of several bacteria, and we believe that these organisms might also be able to utilize MurNAc.

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).

The enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis, were studied in detail with a new enzyme assay. In this assay, the enzyme reaction products were derivatized by reductive coupling to a chromophore, Products mere separated by HPLC and the amount of product was calculated by peak integration, Penta-N-acetylglucosamine (penta-nag) and hexa-N-acetylglucosamine (hexa-nag) mere used as substrates, Hexa-nag was more efficiently converted than penta-nag, which is an indication that hevamine has at least six sugar binding sites in the active site. Tetra-N-acetylglucosamine (tetra-nag) and allosamidin were tested as inhibitors. Allosamidin nas found to be a competitive inhibitor with a K(i) of 3.1 mu M. Under the conditions tested, tetra-nag did not inhibit hevamine, (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10(-9), 2.6 × 10(-7), and 1.8 × 10(-7) M, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.

We report here the cloning and characterization of a cytoplasmic kinase ofClostridium acetobutylicum, named MurK (formurein sugarkinase). The enzyme has a unique specificity for both amino sugars of the bacterial cell wall,N-acetylmuramic acid (MurNAc) andN-acetylglucosamine (GlcNAc), which are phosphorylated at the 6-hydroxyl group. Kinetic analyses revealedKmvalues of 190 and 127 μM for MurNAc and GlcNAc, respectively, and akcatvalue (65.0 s−1) that was 1.5-fold higher for the latter substrate. Neither the non-N-acetylated forms of the cell wall sugars, i.e., glucosamine and/or muramic acid, nor epimeric hexoses or 1,6-anhydro-MurNAc were substrates for the enzyme. MurK displays low overall amino acid sequence identity (24%) with human GlcNAc kinase and is the first characterized bacterial representative of the BcrAD/BadFG-like ATPase family. We propose a role of MurK in the recovery of muropeptides during cell wall rescue inC. acetobutylicum. The kinase was applied for high-sensitive detection of the amino sugars in cell wall preparations by radioactive phosphorylation.

Interaction of 24 different seed lectins/ isolectins from the Leguminosae with muramic acid (MurAc), N-acetylmuramic acid (MurNAc) and muramyl-dipeptides (MDP), was studied by hapten-inhibition of haemagglutination. Although many lectins were shown to interact, irrespective of their monosaccharide-specificity or systematic position, glucose/mannose-specific lectins from the tribe Vicieae exhibited the best affinity for these components of the bacterial cell wall. The discrepancies observed in the binding of the muramyl-dipeptide diastereo-isomers to lectins suggest that the binding is somewhat conformation-dependent. These interactions could be possibly involved in the recognition of bacteria by plants.

MurNAc etherases cleave the unique D-lactyl ether bond of the bacterial cell wall sugar N-acetylmuramic acid (MurNAc). Members of this newly discovered family of enzymes are widely distributed among bacteria and are required to utilize peptidoglycan fragments obtained either from the environment or from the endogenous cell wall (i.e., recycling). MurNAc etherases are strictly dependent on the substrate MurNAc possessing a free reducing end and a phosphoryl group at C6. They carry a single conserved sugar phosphate isomerase/sugar phosphate-binding (SIS) domain to which MurNAc 6-phosphate is bound. Two subunits form an enzymatically active homodimer that structurally resembles the isomerase module of the double-SIS domain protein GlmS, the glucosamine 6-phosphate synthase. Structural comparison provides insights into the two-step lyase-type reaction mechanism of MurNAc etherases: beta-elimination of the D-lactic acid substituent proceeds through a 2,3-unsaturated sugar intermediate to which water is subsequently added.

Lytic transglycosylases cleave the β-(1→4)-glycosidic bond in the bacterial cell wall heteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. Based on sequence alignments, Ser216 in Pseudomonas aeruginosa membrane-bound lytic transglycosylase B (MltB) was targeted for replacement with alanine to delineate its role in the enzyme’s mechanism of action. The specific activity of the Ser216→Ala MltB derivative was less than 12% of that for the wild-type enzyme, while its substrate binding affinity remained virtually unaltered. These data are in agreement with a role of Ser216 in orienting the N-acetyl group on MurNAc at the −1 subsite of MltB for its participation in a substrate-assisted mechanism of action.

The ubiquitous bacterial cell wall sugar N-acetylmuramic acid (MurNAc) carries a unique D-lactyl ether substituent at the C3 position. Recently, we proposed an etherase capable of cleaving this lactyl ether to be part of the novel bacterial MurNAc dissimilation pathway (Dahl, U., Jaeger, T., Nguyen, B. T., Sattler, J. M., Mayer, C. (2004) J. Bacteriol. 186, 2385-2392). Here, we report the identification of the first known MurNAc etherase. The encoding gene murQ is located at 55 min on the Escherichia coli chromosome adjacent to murP, the MurNAc-specific phosphotransferase system. A murQ deletion mutant could not grow on MurNAc as the sole source of carbon and energy but could be complemented by expressing murQ from a plasmid. The mutant had no obvious phenotype when grown on different carbon sources but accumulated MurNAc 6-phosphate at millimolar concentrations from externally supplied MurNAc. Purified MurQ-His6 fusion protein and extracts of cells expressing murQ both catalyze the cleavage of MurNAc 6-phosphate, with GlcNAc 6-phosphate and D-lactate being the primary products. The 18O label from enriched water is incorporated into the sugar molecule, showing that the C3-O bond is cleaved and reformed by the enzyme. Moreover, an intermediate was detected and identified as an unsaturated sugar molecule. Based on this observation, we suggested a lyase-type mechanism (beta-elimination/hydration) for the cleavage of the lactyl ether bond of MurNAc 6-phosphate. Close homologs of murQ were found on the chromosome of several bacteria, and amino acid sequence similarity with the N-terminal domain of human glucokinase-regulatory protein (GckR or GKRP) was recognized.

The inhibitory potency of both muramic acid (MurAc) and N-acetylmuramic acid (MurNAc) on various legume lectins, including Glc/Man- and Gal/GalNAc-specific lectins, was investigated by a haemagglutination inhibition technique. Data indicated that many lectins, especially those specific for Glc/Man, specifically interact with MurAc and MurNAc often to a greater extent than with other monosaccharides and their derivatives, such as N-acetylglucosamine (GlcNAc) and sialic acid. Glc/Man-specific lectins were also shown to interact with the muramyl-dipeptide MurNAc-D-Ala-D-isoGln. These interactions could explain why various lectins readily agglutinate some bacterial strains or which cell walls contain peptidoglycans with high amounts of MurNAc.

In a previous communication (J. Biol. Chem. (1975) 250, 1676-1682), methods were described for the purification of the N-acetylmuramic acid L-alanine amidase from Bacillus subtilis ATCC 6051 and of a modifier protein which combines stoichiometrically with the enzyme and stimulates the activity approximately 3-fold. A detailed examination of the wall cleavage products obtained in the absence and in the presence of modifier indicates that the major effect of the modifier is not to change the enzyme velocity, but rather to change the pattern of cleavage from a more random pattern, when enzyme alone hydrolyzes the cell wall, to a sequential pattern in the presence of modifier protein. Tight binding of the enzyme to the cell wall and functional interaction with the modifier occur only when cell walls from Bacillus subtilis ATCC 6051 containing or teichoic acid are used as a substrate. We suggest that a general function of cell wall teichoic acids is to act as specific "allosteric" ligands for bacterial cell wall lytic enzymes as has been demonstrated previously in Pneumococci (Tomasz, A., and Westphal, M (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 2627-2630).

The N-acetymuramic acid L-alanine amidase from Bacillus subtilis (ATCC 6051) has been purified to homogeneity. It is a monomeric protein of molecular weight 50,000. The enzyme has a high affinity for homologous cell walls and once attached to a cell wall will hydrolyze the wall completely before initiating the hydrolysis of a new cell wall. The affinity of the enzyme for cell walls devoid of teichoic acid or for cell walls of Bacillus megaterium is much lower than that for B. subtilis cell walls. A second homogenous protein has been isolated from B. subtilis which specifically combines with the amidase in a 1:1 molar ratio and stimulates enzyme activity. This modifier protein has no intrinsic cell wall lytic activity. The binding of enzyme and modifier protein has a dissociation constant of 8.5 times 10-9 M in 0.1 M LiCl, pH 8.0, but the two proteins can be completely dissociated in 3 M LiCl at pH 8.0.

The cell subpopulations in the haemolymph of Planorbarius corneus were distinguished by means of flow cytometry. An antibody against N-acetylmuramic acid was prepared and used as a cellular marker to recognize the cell types forming the subpopulations. The spreading haemocytes showed a positive reaction for anti-N-acetylmuramic acid; round haemocytes gave a negative reaction.

Phosphoenolpyruvate:uridine-5'-diphospho-N-acetyl-2-amino-2-deoxyglucose-3-enolpyruvyltransferase, an enzyme which transfers enolpyruvate from phosphoenolpyruvate to uridine diphospho-N-acetylglucosamine (UDP-GlcNAc) with the liberation of inorganic orthophosphate was purified 326-fold from Enterobacter cloacae NRC 492. The transferase was free of UDP-GlcNAc-enolpyruvate reductase, an enzyme which catalyzes the reduction of UDP-GlcNAc-enolpyruvate to form uridine-5'-diphospho-N-acetylmuramic acid. The enzyme was specific for the indicated substrates, and the optimum pH range for the reaction was from pH 6.7 to pH 7.4. The reaction was reversible as judged by radioactive inorganic orthophosphate exchange. The Km for phosphoenolpyruvate was 3 x 10-5 m and that for UDP-GlcNAc was 4.6 x 10-4 m. The available data suggest that the enzyme requires thiol group(s) for its activity. The energy of activation of the enzyme, calculated from the Arrhenius plot, was 15 kcal per mole. Pure UDP-GlcNAc-enolpyruvate has been prepared in milligram quantities. The hydrolysis of this nucleotide in 1 m HCl at 100° liberated 1 mole of pyruvate per mole of uridine. Hydrogenation of the nucleotide followed by hydrolysis in 6 n HCl at 100° resulted in the liberation of muramic acid. On the basis of its chemical properties and infrared and ultraviolet absorption studies, the nucleotide has been characterized as uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-enolpyruvylglucose. Infrared and ultraviolet absorption spectra are given.

Uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucose:NADP-oxidoreductase, an enzyme which catalyzes the reduction of UDP-GlcNAc-enolpyruvate to form uridine-5'-diphospho-N-acetylmuramic acid, was further purified to 1520-fold purification from Enterobacter cloacae NRC 492. Its molecular weight was estimated to be 35,500 by Sephadex gel filtration method. The reductase was greatly stimulated by a number of monovalent cations (T1+ g K+ g NH4+ g Rb+ >> Li+ ≃ Na+). The activation by K+ gave a normal hyperbolic saturation curve with a Ka of 5.8 x 10-3 m. Hill plots suggested a single binding site for K+ in association with each catalytic center. The activation by K+ was instantaneous and did not cause any shift in the pH optimum. The increase in K+ concentration from 2 mm to 20 mm markedly lowered the apparent Km value for UDP-GlcNAc-enolpyruvate. The value changed from 10.5 x 10-6 m to 6.5 x 10-6m. Effect on the Km value for NADPH was small. The data were consistent with the proposal that K+ might be acting as a bridge between UDP-GlcNAc-enolpyruvate and the enzyme.

Uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucose:NADP-oxidoreductase, an enzyme which catalyzes the reduction of UDP-GlcNAc-enolpyruvate to form uridine-5'-diphospho-N-acetylmuramic acid, was purified 151-fold from Enterobacter cloacae NRC 492. The optimum pH range for the reaction was from 8.1 to 8.5. The Km for UDP-GlcNAc-enolpyruvate was 4.0 x 10-5 m and that for NADPH was 4.9 x 10-5 m. The available data suggest that the enzyme requires a thiol group (or groups) for its activity and contains FAD as the prosthetic group.

Isolated cell walls of Bacillus licheniformis ATCC 9945 were digested with lysozyme, and a low molecular weight fraction was obtained after dialysis of the digest. A disaccharide and two disaccharide peptides were isolated from this fraction by gel filtration on a Bio-Gel P-4 column and chromatography on Dowex 50. The structure of the isolated compounds was studied by chemical and enzymatic techniques. The disaccharide was found to be identical with O-2-acetamido-2-deoxy-β-d-glucopyranosyl-(1→4)-2-acetamido-3-O-(d-1-carboxyethyl)-2-deoxy-d-glucose (GlcNAcβ-(1 → 4)MurNAc) previously isolated from Micrococcus lysodeikticus cell walls. The two disaccharide peptides were shown to be tripeptide derivatives of this disaccharide, wherein the peptide is linked to the disaccharide by an amide bond between the carboxyl group of the muramic acid of the latter and the NH2-terminal amino group of the peptide. One of the disaccharide peptides was shown to have the structure [see PDF for structure] (shown in Fig. 4). The other has the same structure as this, but in addition contains an amide group on the α-COOH of glutamic acid. Small amounts of a tetrasaccharide, GlcNAcβ-(1 → 4)MurNAcβ(1 → 4)GlcNacβ(1 → 4)MurNAc, were also isolated.

The association constants with lysozyme of several saccharides containing N-acetyl-d-glucosamine and N-acetylmuramic acid, have been determined by fluorescence spectroscopy or, in one case, by equilibrium dialysis experiments. The association data may be understood on the assumption that the active site of lysozyme consists of a number of subsites binding individual 2-acetamido-2-deoxyglucopyranose rings. On the basis of these data, we have drawn detailed conclusions about the specificity of the individual subsites and their contributions to the total binding energy of oligosaccharide substrates and inhibitors. These conclusions are consistent with, and offer further support of, the mechanism of lysozyme action deduced by Phillips and his coworkers on the basis of x-ray crystallographic studies.

Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. In this paper the cleavage specificity of hevamine for peptidoglycan was studied by HPLC and mass-spectrometry analysis of enzymatic digests. The results clearly showed that the enzyme cleaves between the C-1 of a N-acetylglucosamine and the C-4 of a N-acetylmuramate residue. This means that hevamine, and very likely also other family 18 glycosyl hydrolases which cleave peptidoglycan, cannot be classified as lysozymes. (C) 1997 Federation of European Biochemical Societies.

The N-acetylmuramic acid L-alanine amidase from Bacillus subtilis W-23 has been purified to apparent homogeneity. The enzyme is a monomer of molecular weight 51,000, which binds extremely tightly to homologous cell walls but not to heterologous cell walls, even of the closely related strain B. subtilis ATCC 6051. This difference in binding is only in part due to differences in teichoic acid between these two strains and to a large extent appears to represent differences in the arrangement of the peptidoglycan. A comparison of the amidase from B. subtilis W-23 and the enzyme previously purified from B. subtilis ATCC 6051 (Herbold and Glaser, 1975) shows that the two proteins, which cleave the same bond and are of the same size, do not cross-react immunologically and that the two enzymes are, therefore, not closely related in structure.

The purification of the N-acetylmuramic acid l-alanine amidase from Bacillus megaterium KM is described. The enzyme has a molecular weight of 20,000 by sucrose density centrifugation and by acrylamide gel electrophoresis in sodium dodecyl sulfate. The isoelectric point is 8.2. The enzyme has a pH optimum of 6.8 and a sharp ionic strength optimum at Γ/2 = 0.02. The enzyme has a 4- to 10-fold higher initial velocity with cell walls obtained from logarithmically growing cells than from cell walls obtained from cells in stationary phase of growth. This difference in hydrolysis rate may account, in part, for the difference in cell wall turnover previously observed between stationary and logarithmic cells.