Is Muramidase-2 of Enterococcus hirae a Penicillin-Binding Protein?
Highly purified muramidase-2 (M-2), obtained from broken cell preparations of Enterococcus hirae (Streptococcus faecium) ATCC 9790 was shown to bind penicillin G (Pen G) with low affinity (Dolinger et al., 1989). Fluorography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of [3H]Pen-labeled, purified M-2 showed the presence of two radioactive bands, at about 125 and 70 kDa, both of which were shown to possess peptidoglycan hydrolase activity after elution and renaturation of the protein bands. The electrophoretic mobility of the 70-kDa band was indistinguishable from that of penicillin-binding protein (PBP) 5 of membrane preparations of E. hirae. More recently, immunochemical and molecular studies clearly demonstrated that PBP 5 and the 70-kDa form of M-2 were separate and distinct proteins (Shockman et al., these proceedings; Tepper et al., in preparation). Because penicillin binding appears to be closely associated with penicillin-interactive, active-site serine proteins, such as β-lactamases, transpeptidases and carboxypeptidases (Ghuysen, 1991), binding to a muramidase seemed unusual. However, the derived amino acid sequence of cloned M-2 showed the presence of an SXXK sequence (S175NTK), as well as other amino acid motifs characteristic of penicillin-interactive proteins (Joris et al., 1992; Shockman et al., these proceedings). Recent biochemical studies of M-2 (Chu et al., 1992; Kariyama and Shockman, 1992) made it possible to analyze penicillin binding to the extracellular form of M-2, decreasing the possibility of contamination of M-2 with membrane and cell wall proteins.
KeywordsHuman Serum Albumin Ammonium Sulfate Enzyme Complex Cell Wall Protein Amino Acid Motif
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