Browsing publications of the junior research group chemical biology of carbohydrates ([HIPS]CBCH) by Authors
An efficient synthesis of 1,6-anhydro- N -acetylmuramic acid from N -acetylglucosamineCalvert, Matthew B; Mayer, Christoph; Titz, Alexander; Helmholtz Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-12-11)
The exo-β-N-acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases.Müller, Maraike; Calvert, Matthew; Hottmann, Isabel; Kluj, Robert Maria; Teufel, Tim; Balbuchta, Katja; Engelbrecht, Alicia; Selim, Khaled A; Xu, Qingping; Borisova, Marina; et al. (Elsevier, 2021-03-05)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.
N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral PathogenTannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism.Hottmann, Isabel; Mayer, Valentina M T; Tomek, Markus B; Friedrich, Valentin; Calvert, Matthew B; Titz, Alexander; Schäffer, Christina; Mayer, Christoph; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2018)Tannerella forsythia is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugarN-acetylmuramic acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of theEscherichia colietherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described forT. forsythia.In between the respective genes on theT. forsythiagenome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 104-fold higher catalytic efficiency (kcat/Km) for MurNAc than forN-acetylglucosamine (GlcNAc) withkcatvalues of 10.5 s-1and 0.1 s-1andKmvalues of 200 μM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition (Ki[S]= 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism ofT. forsythia, a kinase deletion mutant (ΔTf_murK::erm) was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, theΔTf_murK::ermmutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 μg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 μg/ml MurNAc for optimal growth. These data reveal thatT. forsythiareadily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion ofTf_murKblocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of theT. forsythiacell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis.