Structure-function studies towards the mechanisms of exo-lytic N-acetylmuramidases of CAZy families GH170 and GH171

Lysozyme-like endo-N-acetylmuramidases, which catalyze the endo-lytic cleavage of the peptidoglycan cell wall of bacteria, are intensively studied and well-characterized antimicrobial enzymes. In contrast, little is known about exo-β-N-acetylmuramidases that catalyze the exo-lytic cleavage of N-acetylmuramic acid (MurNAc) and 6-phospho-MurNAc entities from non-reducing termini. We recently identified such enzymes constituting two novel families of glycosyl hydrolases, GH171 (www.cazy.org/GH171.html) and GH170 (www.cazy.org/GH170.html), respectively. NamZ of B. subtilis (BsNamZ)¹ and two related enzymes from Tannerella forsythia (TfNamZ1 and TfNamZ2)² were identified and classified into GH171, whereas MupG of Staphylococcus aureus (SaMupG)³ and MupG-like enzymes from Priestia megaterium (i.e. Bacillus megaterium) and Lactobacillus pasteurii were identified and classified into GH170. Chemical synthesis of chromogenic substrates facilitated the identification and initial characterization of these enzymes and they are now used, in combination with x-ray crystallography, mutagenesis and kinetic studies of active site variants, to elucidate the structure, function and mechanism of these enzymes. We are presenting structural and kinetic data that shine a light on the mechanisms of these unique exo-lytic muramidases.

¹ Müller M, Calvert C, Hottmann I, Kluj RM, Teufel T, Balbuchta K, Engelbrecht A, Selim KA, Xu Q, Borisova M, Titz A, Mayer C (2021) The exo-β-N-acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases, J Biol Chem. 296: 100519.
² Borisova M, Balbuchta K, Lovering A, Titz A, Mayer C. (2022) NamZ1 and NamZ2 from the oral pathogen Tannerella forsythia are peptidoglycan processing exo-β-N-acetylmuramidases with distinct substrate specificities. J Bacteriol. 2022 Mar 15;204(3):e0059721.
³ Kluj RM, Ebner P, Adamek M, Ziemert N, Mayer C, Borisova M. (2018) Recovery of the peptidoglycan turnover product released by the autolysin Atl in Staphylococcus aureus involves the phosphotransferase system transporter MurP and the novel 6-phospho-N-acetylmuramidase MupG. Front Microbiol. 9:2725.