The mature forms of the extracellular muramidase-2 of Enterococcus hirae and Streptococcus faecalis autolysin have very similar primary structures. Each consists of an active-site-containing N-terminal domain fused to a multiple-repeat C-terminal domain. Polypeptide segments occurring at equivalent places in these two bacterial wall lytic enzymes have homologues in two phage lysozymes and in three functionally unrelated proteins, illustrating the principle that protein molecules frequently are constructed from modules that are linked in a single polypeptide chain.

Extracellular muramidase-2 of Enterococcus hirae ATCC 9790 was purified to homogeneity by substrate binding, guanidine-HCI extraction, and reversed-phase chromatography. A monoclonal antibody, 2F8, which specifically recognizes muramidase-2, was used to screen a genomic library of E. hirae ATCC 9790 DNA in bacteriophage Agtll. A positive phage clone containing a 4.5-kb DNA insert was isolated and analyzed. The EcoRI-digested 4.5-kb fragment was cut into 2.3-, 1.0-, and 1.5-kb pieces by using restriction enzymes KpnI, Sau3AI, and PstI, and each fragment was subcloned into plasmid pJDC9 or pUC19. The Two muramidases, muramidase-1 and muramidase-2, have been found in Enterococcus hirae ATCC 9790 (formerly Streptococcus faecium) (15). Muramidase-1 is a glycoprotein (16) which can hydrolyze the re-N-acetylated peptidoglycan and the sodium dodecyl sulfate (SDS)-treated cell walls of E. hirae ATCC 9790 (15). Muramidase-1 is present in a latent form (130 kDa) that can be converted into an active form (87 kDa) via the action of any of a variety of proteinases, including trypsin (16). Muramidase-1 is unusual in that it possesses about 12 5-mercaptouridine monophosphate residues covalently linked to the protein (6).Muramidase-2 is also an unusual enzyme that appears to be a gene product different from muramidase-1 (23). It differs from muramidase-1 in substrate specificity and rapidly hydrolyzes the nonacetylated peptidoglycan fraction of E. hirae and walls of Micrococcus luteus (15), both of which are resistant to the action of muramidase-1. When extracted from intact or disrupted bacteria, highly purified preparations of muramidase-2, upon SDS-polyacrylamide gel electrophoresis (PAGE), showed the presence of two polypeptides of about 125 and 74 kDa (5), both of which possess peptidoglycan hydrolase activity. Substantial amounts of the smaller, 74-kDa form have been found in and isolated from the culture medium (14,15 (Irvine, Calif.). The isopropyl-,-D-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-,-D-galactoside (X-Gal) were purchased from Bethesda Research Laboratories.Purification of extracellular muramidase-2. Cultures of E. hirae were grown to early stationary phase (1.3 mg [dry weight] per ml) in 600 ml of S broth (14). The culture was rapidly chilled to 0°C and centrifuged (20,000 x g, 10 min, 4°C). Sonicated E. hirae peptidoglycan (50 mg in 5 ml of 10 mM sodium phosphate [pH 7.0]) was added to the supernatant culture medium, and the mixture was allowed to stand at 0°C for 30 min with occasional mixing. After binding, the peptidoglycan-enzyme complex was pelleted (20,000 x g, 20 min, 4C); the pellet was suspended in 1 ml of 10 mM sodium phosphate, pH 7.0, transferred into a microcentrifuge tube, centrifuged (14,000 x g, 15 min, 4C), resuspended, and then washed in 1.5 ml of 10 mM sodium phosphate, pH 7.0. The pellet was extracted twice with 500 ,ul of 6 M guanidine-HCl, and the extracts were combined and then stored at -70°C. For further purification, defrosted 6 M guanidine-HCl extract was inject...

The mature forms of the extracellular muramidase‐2 of Enterococcus hirae and Streptococcus faecalis autolysin have very similar primary structures. Each consists of an active‐site‐containing N‐terminal domain fused to a multiple‐repeat C‐terminal domain. Polypeptide segments occurring at equivalent places in these two bacterial wall lytic enzymes have homologues in two phage lysozymes and in three functionally unrelated proteins, illustrating the principle that protein molecules frequently are constructed from modules that are linked in a single polypeptide chain.

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