Exo-β-N-acetylmuramidase NamZ ofBacillus subtilisis 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 potentially lyse bacterial cells. They catalyze an endo-lytic cleavage of the peptidoglycan, the structural component of the bacterial cell wall; i.e. they hydrolyze glycosidic N-acetylmuramic acid (MurNAc)-β-1,4-N-acetylglucosamine (GlcNAc)-bonds within the heteroglycan backbone of peptidoglycan. In contrast, little is known about exo-β-N-acetylmuramidases, catalyzing 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 identified ybbC of B. subtilis, renamed namZ, as encoding 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. 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. NamZ is a member of the DUF1343 protein family of unknown function and shows no significant sequence identity with known glycosidases. A structural model of NamZ revealed a putative active site located in a cleft within the interface of two subdomains, one of which constituting a Rossmann-fold-like domain, unusual for glycosidases. On this basis, we propose that NamZ represents the founding member of a novel family of peptidoglycan hexosaminidases, which is mainly present in the phylum Bacteroidetes and, less frequently, within Firmicutes (Bacilli, Clostridia), Actinobacteria and Gammaproteobacteria.

NamZ1 and NamZ2 from the oral pathogen Tannerella forsythia are peptidoglycan processing exo-β-N-acetylmuramidases with distinct substrate specificity

“…Here we present the characterization of two of them and show that, despite of their high amino acid sequence identity, TfNamZ1 and TfNamZ2 differ in their substrate specificity and product formation. TfNamZ1 is an exo-β- N -acetylmuramidase that releases primarily GlcNAc-MurNAc from the non-reducing end of peptide-free PGN glycan strands, whereas TfNamZ2 releases MurNAc from the non-reducing ends of the glycans and thus, possesses classical exo-β- N -acetylmuramidase activity, similar to that previously described for BsNamZ [Müller, 2021 #18933]. Interestingly, TfNamZ1 is encoded within a putative operon in the T. forsythia genome that also contains the genes encoding TfMurT and TfAmpG, as well as a number of other enzymes related to PGN salvage (Fig.…”

“…The process of PGN turnover and utilization as a nutrient and energy source (i.e., PGN recycling or salvage), also involves the activity of exo-lytic hydrolases, which processively degrade PGN from the ends, thereby averting uncontrolled autolysis [Litzinger, 2010 #12434]. We previously showed that Bacillus subtilis is able to digest intact PGN by sequential hydrolysis from the non-reducing ends via the joined activity of the exo-β- N -acetylglucosamidase BsNagZ, the exo- N -acetylmuramoyl-L-alanine amidase AmiE, and the exo-lytic N -acetylmuramidase BsNamZ, which sequentially release GlcNAc, peptides, and MurNAc, respectively [Litzinger, 2010 #12434; Müller, 2021 #18933]. Presumably, these hydrolases are involved in the degradation of PGN during turnover of the cell wall or the decay of lysed cells in a starving population of B. subtilis [Borisova, 2016 #14239].…”

“…The exo-lytic β- N -acetylmuramidase BsNamZ, was recently characterized and shown to constitute a novel family of glycosidases (CAZy GH171; www.cazy.org/GH171.html) [Müller, 2021 #18933]. BsNamZ uniquely cleaves terminal N -acetylmuramic acid (MurNAc) moieties from the non-reducing ends (exo-lytic cleavage) of peptide-free, PGN glycans and also hydrolyzes the artificial substrate 4-nitrophenyl β-MurNAc (pNP-MurNAc).…”

“…We previously recognized that putative NamZ orthologs are narrowly distributed within bacteria and are frequently found within members of the Bacteroidetes phylum [Müller, 2021 #18933]. The oral pathogen Tannerella forsythia , which is strongly associated with disease, such as periodontitis and rheumatoid arthritis, belongs to the Bacteroidetes [Loyola-Rodriguez, 2010 #19862;Martinez-Rivera, 2017 #19873;Bourgeois, 2019 #19864].…”

“…The namZ operon of B. subtilis strain 168 (NCBI Reference Sequence accession no. NC_000964.3) includes three genes ( amiE , nagZ and namZ ) involved in PGN turnover/recycling and three genes ( murQRP ) required for the uptake of MurNAc and the transcriptional regulation of the operon: amiE encodes the exo-MurNAc-L-Ala amidase BsAmiE [Litzinger, 2010 #12434], nagZ encodes the exo-β- N -acetylglucosaminidase BsNagZ [Litzinger, 2010 #12680] and namZ encodes the recently identified and characterized exo-β- N -acetylglucosaminidase BsNamZ (in red) [Müller, 2021 #18933]; furthermore, murP encodes the MurNAc-specific phosphotransferase system (PTS) transporter BsMurP, murQ encodes the MurNAc 6-phosphate etherase MurQ and murR encodes the putative transcriptional regulator of the operon [Borisova, 2016 #14239]. Within the genome of T. forsythia strain ATCC 43037 (NCBI Reference Sequence: NZ_JUET00000000.1), three orthologous namZ -like family GH171 glycosidases (CAZy GH171; www.cazy.org) were identified, TfnamZ1 (Tanf_08370) , TfnamZ2 (Tanf_00660) and TfnamZ3 ( Tanf_00855 ).…”