title: Biochemical characterization of the major N-acetylmuramidase from Lactobacillus buchneri.
abstract: Bacterial cell wall hydrolases are essential for peptidoglycan remodelling in regard to bacterial cell growth and division. In this study, peptidoglycan hydrolases (PGHs) of different Lactobacillus buchneri strains were investigated. First, the genome sequence of L. buchneri CD034 and L. buchneri NRRL B-30929 was analysed in silico for the presence of PGHs. Of 23 putative PGHs with different predicted hydrolytic specificities, the glycosyl hydrolase family 25 domain-containing homologues LbGH25B and LbGH25N from L. buchneri CD034 and NRRL B-30929, respectively, were selected and characterized in detail. Zymogram analysis confirmed hydrolysing activity on bacterial cell walls for both enzymes. Subsequent reversed-phase HPLC and MALDI-TOF MS analysis of the peptidoglycan breakdown products from L. buchneri strains CD034 and NRRL B-30929, and from Lactobacillus rhamnosus GG, which served as a reference, revealed that LbGH25B and LbGH25N have N-acetylmuramidase activity. Both enzymes were identified as cell wall-associated proteins by means of immunofluorescence microscopy and cellular fractionation, as well as by the ability of purified recombinant LbGH25B and LbGH25N to bind to L. buchneri cell walls in vitro. Moreover, similar secondary structures mainly composed of β-sheets and nearly identical thermal stabilities with Tm values around 49 °C were found for the two N-acetylmuramidases by far-UV circular dichroism spectroscopy. The functional and structural data obtained are discussed and compared to related PGHs. In this study, a major N-acetylmuramidase from L. buchneri was characterized in detail for the first time.
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology unit, Universität für Bodenkultur Wien, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Julia
authors.initials: J
authors.lastname: Anzengruber
authors.affiliation: AgroParisTech, UMR Micalis, Jouy-en-Josas, France.|INRA and AgroParisTech, UMR1319 Micalis, 78350 Jouy-en-Josas, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Courtin
authors.affiliation: Center of Microbial and Plant Genetics, K.U. Leuven, 3001 Leuven, Belgium.
authors.forename: Ingmar J J
authors.initials: IJJ
authors.lastname: Claes
authors.affiliation: VIBT Imaging Centre, Universität für Bodenkultur Wien, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Monika
authors.initials: M
authors.lastname: Debreczeny
authors.affiliation: Department of Chemistry, Universität für Bodenkultur Wien, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hofbauer
authors.affiliation: Department of Chemistry, Universität für Bodenkultur Wien, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Obinger
authors.affiliation: AgroParisTech, UMR Micalis, Jouy-en-Josas, France.|INRA and AgroParisTech, UMR1319 Micalis, 78350 Jouy-en-Josas, France.
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Chapot-Chartier
authors.affiliation: Center of Microbial and Plant Genetics, K.U. Leuven, 3001 Leuven, Belgium.
authors.forename: Jos
authors.initials: J
authors.lastname: Vanderleyden
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology unit, Universität für Bodenkultur Wien, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Paul
authors.initials: P
authors.lastname: Messner
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology unit, Universität für Bodenkultur Wien, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Christina
authors.initials: C
authors.lastname: Schäffer
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007778:Lactobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D010457:Peptidoglycan / Q000378:metabolism; D017433:Protein Structure, Secondary; D021381:Protein Transport
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; D006026:Glycoside Hydrolases; C026255:endo-N-acetylmuramidase