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. 2010 Oct 27;285(53):41232–41243. doi: 10.1074/jbc.M110.156273

FIGURE 4.

FIGURE 4.

Identification of the critical amino acid residues involved in CwlP muramidase activity. A, alignment of the muramidase domain of CwlP, homologous proteins, lytic transglycosylases, and muramidases. The alignment was performed using the Pfam database. The characterized or predicted critical amino acid residues for the hydrolytic activity (Refs. 19, 33, 34, 35) are shown in boxes. Amino acid residues identical to those in the CwlP sequence are shaded. Numbers indicate the amino acid positions from the N terminus. Gray rectangles and broken arrows denote secondary structures, α-helices, and β-sheets in the Slt70 E. coli protein (SLT_ECOLI) (23, 36). Arrows indicate amino acid residues that were mutated. An asterisk denotes the amino acid that was exchanged and is not conserved between SLT_ECOLI and MltC_ECOLI. Abbreviations: CwlP_SLT, SLT domain (exhibiting CwlP muramidase activity); YjbJ_BACSU, B. subtilis YjbJ protein; YjbJ_BACLI, Bacillus licheniformis YjBJ protein; YjbJ_BACHA, Bacillus halodurans YjbJ protein; Geobacillus, Geobacillus sp. protein; Heliobacterium, Heliobacterium modesticaldum protein; Shewanella, Shewanella benthica protein; Acinetobacter, Acinetobacter baumannii protein; Lawsonia, Lawsonia intracellularis protein; Pelotomaculum, Pelotomaculum thermopropionicum protein; Lysinibacillus, Lysinibacillus sphaericus protein; Oceanobacillus, Oceanobacillus iheyensis protein; Clostridium, Clostridium cellulolyticum protein; Desulfotomaculum, Desulfotomaculum reducens protein; SLT_ECOLI, soluble lytic transglycosylase 70 in E. coli; MltC_ECOLI, membrane-bound lytic murein transglycosylase C in E. coli; LYG_ANSAN, Anser anser anser goose-type lysozyme; CwlT_N, N-terminal domain (muramidase) of CwlT. Panel B, in vitro cell wall hydrolytic activity. Purified cell walls (0.3 mg/ml) were digested with 1 μm (15 μg/ml) CwlP-SLT or the mutated CwlP-SLT for 30 min at 37 °C, pH 5.0. The reduction in the turbidity of the solution for the mutated CwlP-SLT was compared with that of the wild-type CwlP-SLT. In this experiment, the mutated residues E1447Q and E1447A exhibited no activity toward B. subtilis cell walls.