The exo-β-N-acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases

Abstract

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.

Keywords: N-acetylglucosaminidase; N-acetylmuramidase; N-acetylmuramoyl amidase; Rossmann-fold; cell wall recycling; exo-lytic glycosidase; lysozyme; peptidoglycan hydrolase.

Figure 1

Accumulation of specific cell wall fragments in growth supernatants of B. subtilis WT, ΔnamZ, and ΔnagZ cells. Supernatants of stationary phase cultures (grown for 20 h in LB) of B. subtilis WT (A), ΔnamZ, (B) and ΔnagZ (C) were analyzed by LC-MS for the accumulation of small cell wall fragments. Shown are the base peak chromatograms (BPCs; black) and extracted ion chromatograms (EIC) for the disaccharides MurNAc-GlcNAc (gray), GlcNAc-MurNAc (brown), GlcNAc-anhMurNAc (orange) as well as anhMurNAc (light blue) and the trisaccharide MurNAc-GlcNAc-anhMurNAc (green). To unequivocally identify the accumulation products, these were treated with recombinant NagZ (8).

Figure 2

NamZ of B. subtilis specifically cleaves the chromogenic substrate pNP-MurNAc. The other tested chromogenic substrates, pNP-GlcNAc and oNP-Gal were not cleaved by NamZ, but are substrates of the exo-β-N-acetylglucosaminidase of B. subtilis (NagZ) and the β-d-galactosidase of E. coli (LacZ), respectively. Purified recombinant NamZ and NagZ from B. subtilis, as well as commercial LacZ (100 μg/ml each) were incubated for 30 min with the indicated chromogenic compound (100 μM each). Color intensity was enhanced by adding an equal volume of sodium borate buffer (100 mM, pH 10).

Figure 3

Determination of the kinetic parameters of NamZ using the chromogenic substrate pNP-MurNAc at pH 8.0 and MurNAc-GlcNAc substrate at pH 7.0.A, kinetic parameters using the chromogenic substrate pNP-MurNAc were determined as described in Experimental procedures. B, standard curve for 4-nitrophenol (13–430 μM) was determined in 0.2 M phosphate buffer (pH 8.0) (black squares). Absorption of pNP was measured at 405 nm. For all experiments, standard errors (SEM) are indicated and calculated out of three biological replicates. Standard curve for MurNAc (6.25–800 μM) was determined in 0.2 M phosphate buffer (pH 7.0) and stopping buffer (1% formic acid, 0.5% ammonium formate, pH 3.2) in equal amounts in a total volume of 50 μl. Samples were analyzed using HPLC-MS and the areas under the curve (AUCs) were generated using the extracted ion chromatograms (EICs) intensities for MurNAc ((M-H)⁻ 292.113 m/z) (gray circles). C, kinetic parameters using purified MurNAc-GlcNAc were determined. Standard errors (SEM) are indicated and calculated out of three biological replicates. D, kinetic parameters using purified MurNAc-GlcNAc as substrate were determined for substrate concentrations from 0.025 to 0.2 mM as described in Experimental procedures. Standard errors (SEM) are indicated and calculated out of three biological replicates.

Figure 4

Sequential digest of peptidoglycan by NagZ, NamZ, and AmiE. Purified peptidoglycan was sequentially digested by the action of NagZ, NamZ, and AmiE and analyzed by HPLC-MS. Main products are shown as extracted ion chromatograms (EIC). Single cell wall carbohydrates could be identified with observed masses (A) (M + H)⁺ 222.098 m/z for GlcNAc (red) with a retention time of 10.1 min, (B) (M-H)⁻ 292.104 m/z for MurNAc (dark blue) with a retention time of 21.7 min and (M-H)⁻ 274.094 m/z for anhMurNAc (light blue) with a retention time of 24.8 min. C, peptides could be identified with observed masses (M-H)⁻ 388.187 m/z for tripeptide with an amidation (black) with a retention time of 8.5 min, (M-H)⁻ 830.406 m/z for tri-tetrapeptide with two amidations (dark gray) with a retention time of 12.1 min, and (M-H)⁻ 831.388 m/z for tri-tetrapeptide with one amidation (light gray) with a retention time of 15.1 min.

Figure 5

A structure model of B. subtilis NamZ depicts a novel glycosidase-fold. A structural model of NamZ, constructed by HHPred, reveals a putative active site located in a cleft within the interface of two subdomains. The N-terminal catalytic domain contains a Rossmann-fold-like domain (lower part) and contains a conserved glutamate (Glu92) at the tip of the second β-strand. The C-terminal auxiliary domain contains a highly exposed conserved arginine (Arg279).

Figure 6

Phylogenetic tree showing the distribution of peptidoglycan recycling associated proteins in representative bacterial taxa. MurQ (blue) is a representative enzyme of the MurQ pathway present in E. coli and B. subtilis (16, 21, 22), and MupG (green) is a representative of the modified MurQ pathway identified in S. aureus (15). AmgK (gray), MupP (purple), and MurU (teal) are representatives of an alternative, “anabolic” recycling pathway described in P. putida (35). NagZ (orange) and NamZ (red) pinpoint the putative presence of autolytic pathways described in B. subtilis (16). The tree is built on the taxonomy according to NCBI and MultiGeneBlast searches for these proteins, using the studies by (35, 50) as reference.

Figure 7

Overview of the roles of NagZ, AmiE, and NamZ of the peptidoglycan recycling/MurNAc catabolic operon in the degradation of peptidoglycan in B. subtilis. The exo-lytic peptidoglycan hydrolases NagZ (exo-β-N-acetylglucosaminidase), AmiE (exo-N-acetylmuramyl-l-alanine amidase), and NamZ (formerly YbbC; exo-β-N-acetylmuramidase) cleave-off GlcNAc, peptides, and MurNAc residues, respectively, from the nonreducing terminus of peptidoglycan chains by sequential reactions, as indicated (1. NagZ, 2. AmiE, and 3. NamZ). Furthermore, these enzymes may act on shorter fragments released from the peptidoglycan by the action of endo-lytic and disaccharide-releasing exo-lytic autolysins. Apparently, NamZ primarily cleaves MurNAc-GlcNAc and MurNAc-GlcNAc-anhMurNAc, whereas NagZ cleaves GlcNAc-anhMurNAc and GlcNAc-MurNAc. Thereby released monosaccharides, MurNAc and GlcNAc, are recovered by B. subtilis via the phosphotransferase system transporters MurP and NagP, yielding in the cytoplasm MurNAc 6-phosphate (MurNAc-6P) and GlcNAc 6-phosphate (GlcNAc-6P), respectively. The MurNAc-6P lactyl ether hydrolase (etherase) MurQ converts MurNAc-6P to GlcNAc-6P in the cytoplasm, which enters pathways leading to either cell wall synthesis or glycolysis.