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17 p.-7 fig.-2 tab. Rico-Lastres, Palma et al., Streptococcus pneumoniae is a major cause of life-threatening diseases worldwide. Here we provide an in-depth functional characterization of LytB, the peptidoglycan hydrolase responsible for physical separation of daughter cells. Identified herein as an N-acetylglucosaminidase, LytB is involved also in colonization and invasion of the nasopharynx, biofilm formation and evasion of host immunity as previously demonstrated. We have shown that LytB cleaves the GlcNAc-β-(1,4)-MurNAc glycosidic bond of peptidoglycan building units. The hydrolysis occurs at sites with fully acetylated GlcNAc moieties, with preference for uncross-linked muropeptides. The necessity of GlcN acetylation and the presence of a single acidic moiety (Glu585) essential for catalysis strongly suggest a substrate-assisted mechanism with anchimeric assistance of the acetamido group of GlcNAc moieties. Additionally, modelling of the catalytic region bound to a hexasaccharide tripentapeptide provided insights into substrate-binding subsites and peptidoglycan recognition. Besides, cell-wall digestion products and solubilisation rates might indicate a tight control of LytB activity to prevent unrestrained breakdown of the cell wall. Choline-independent localization at the poles of the cell, mediated by the choline-binding domain, peptidoglycan modification, and choline-mediated (lipo) teichoic-acid attachment contribute to the high selectivity of LytB. Moreover, so far unknown chitin hydrolase and glycosyltransferase activities were detected using GlcNAc oligomers as substrate., Research was funded by grants from the Ministerio de Ciencia e Innovación (MICINN) and the Ministerio de Economía y Competitividad (MINECO) to P. García (SAF2009-10824 and SAF2012-39444-C02-01) and M. Menéndez (BFU2009-10052 and BFU2012-36825), the Consejería de Educación de la Comunidad de Madrid (S2010/BMD/2457) to M. Menéndez. The work in the United Kingdom and the US was supported by grants from the BBSRC (BB/G015902/1) to W. Vollmer and from the National Institutes of Health (GM61629) to S. Mobashery. Additional funding was provided by the CIBER de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII). Palma Rico-Lastres and Roberto Díez-Martínez were the recipients of fellowships from the MICINN (FPI program).