The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin

Abstract

A second peptidoglycan hydrolase (muramidase-2) of Streptococcus faecium ATCC 9790 (Enterococcus hirae) has been purified to apparent homogeneity. The enzyme has been shown to be a 0-1,4-N-acetylmuramoylhydrolase (muramidase; EC 3.2.1.17) and to differ in substrate specificity from a previously isolated muramidase. Purified enzyme appears as two protein staining bands with molecular masses of 125 and 75 kilodaltons (kDa) on polyacrylamide gels after sodium dodecyl sulfate electrophoresis. Elution and renaturation of protein bands from sodium dodecyl sulfate-polyacrylamide gels showed that both proteins have muramidase-2 activity. Both proteins have been shown to bind radioactive benzylpenicillin and have the same electrophoretic mobilities as penicillin-binding proteins 1 and 5 present in membrane preparations of this organism, respectively. Incubation of a ['4C]penicillin G-labeled 125-kDa form of the enzyme with crude alkaline extracts from S. faecium (which did not contain added proteinase inhibitors) showed the endogenous conversion of the radiolabeled 125-kDa form to the radiolabeled 75-kDa form of the enzyme.Streptococcus faecium ATCC 9790 (Enterococcius hirae [10]) was previously shown to possess two distinct peptidoglycan hydrolases, which differ from each other in substrate specificity, molecular mass, and, very probably, mechanism of hydrolysis (19). Muramidase-1 was isolated and purified to homogeneity by affinity chromatography on concanavalin A-Sepharose and was shown to occur in a latent 130-kilodalton (kDa) form that can be proteolytically converted to an active 87-kDa form (20, 24). Muramidase-1 was also shown to contain covalently attached monomeric and oligomeric glucose (20) and monomeric 5-mercaptouridine monophosphate (8). In addition, muramidase-1 was shown to processively hydrolyze linear-soluble, un-crosslinked peptidoglycan chains (1).A second peptidoglycan hydrolase (muramidase-2) has been partially purified from culture supernatants by affinity binding to, and elution from, sodium dodecyl sulfate (SDS)-treated cell walls of Micrococcus luteus and has been shown to be a . It was shown to differ from muramidase-1 in substrate specificity, dissolving the cell walls of M. luteus or the acid-insoluble peptidoglycan fraction of S. faecium walls but having little ability to dissolve S. faecium cell walls. Additional data suggest that muramidase-1 and muramidase-2 are separate gene products.We have purified muramidase-2 from the insoluble pellet of disrupted S. faecium cells. Muramidase-2 was extracted with 0.02 N NaOH at 0°C, bound to the acid-insoluble peptidoglycan fraction of S. faecium, and reextracted with alkali. SDS-polyacrylamide gel electrophoresis (PAGE) of fractions highly enriched for muramidase-2 activity showed the presence of two protein bands, at 125 and 75 kDa. Upon elution and renaturation from SDS gels, both protein bands were shown to possess muramidase-2 activity. The two proteins appeared to be biochemically and immunochemically related and may represent different forms ...

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

“…E. hirae was grown in a complex medium (S broth [5] (7,16). SDS-washed cell walls of E. hirae and M. luteus and acid-treated and re-N-acetylated peptidoglycan of E. hirae were prepared as described previously (16).…”

“…Two polypeptide bands, one of about 125 kDa and the other of about 71 kDa, were shown to possess M-2 activity. Both polypeptides bind radioactive penicillin with low affinity, and evidence was obtained that the 71-kDa form was derived from the 125-kDa form (7). M-2 differs from M-1 in several properties, including substrate specificity.…”

“…M-2 was also purified to apparent homogeneity from the insoluble pellet of disrupted E. hirae (7). Two polypeptide bands, one of about 125 kDa and the other of about 71 kDa, were shown to possess M-2 activity.…”

“…The presence in E. hirae of two apparently distinct muramidases, both of which appeared to be present in unfolded or partially folded forms in addition to active forms (7,8,17,18,29), resulted in the need to efficiently extract and concentrate both activities, either together or separately, and to consider the stability, yield, and cellular locations of each enzyme.…”

The Problems Associated with Enzyme Purification

“…According to Shockman et al (1967), the treatment of the cell wall by sodium dodecyl sulfate (SDS) would resolve the problem of the insolubility of the autolysins of lactic bacteria during purification. SDS is the most widely used detergent to solubilize most otherwise insoluble proteins such as autolysins (Dolinger et al, 1989), its presence may affect the tertiary structure of proteins because it is one of the most strongly denaturing detergents. It is also one of the most difficult to remove with dialysis.…”