Bacteriophage T7 Lysozyme Is an N-Acetylmuramyl-l-alanine Amidase

Abstract

Abstract Studies on the specificity of a lytic enzyme induced by bacteriophage T7 infection of Escherichia coli were carried out by analyzing the products of the enzyme's action on purified E. coli peptidoglycan. The E. coli peptidoglycan is known to contain a lipoprotein (molecular weight = 7,200) covalently attached to diaminopimelic acid. When radioactively labeled E. coli peptidoglycan was digested with purified bacteriophage T4 lysozyme, the lipoprotein was cleaved from the peptidoglycan and migrated to a position of molecular weight 10,600 in a sodium dodecyl sulfate polyacrylamide gel electrophoretogram. The lipoprotein was found to contain both radioactive N-acetylglucosamine and diaminopimelic acid, as expected from the known enzymatic specificity of T4 lysozyme (an N-acetylmuramidase). On the other hand, when the labeled peptidoglycan was digested with a bacteriophage T7 lysate, the lipoprotein migrated to a position of significantly lower molecular weight than that of the lipoprotein cleaved by T4 lysozyme. Furthermore, the lipoprotein contained diaminopimelic acid, but not N-acetylglucosamine. This suggested that the release of the lipoprotein from the peptidoglycan by the T7 lysate was not due to the hydrolysis of one of the bonds in the glycan backbone, but due to the hydrolysis of one of the bonds in the peptide portion of the peptidoglycan. Peptidoglycan radioactively labeled with l-alanine was prepared, digested with the lytic enzyme in a T7 lysate, and the digestion products analyzed. From these experiments, it was concluded that the T7 enzyme cleaves the bond between N-acetylmuramic acid and l-alanine. This conclusion was confirmed by observing that the release of NH2-terminal l-[14C]alanine was chemically detected by dinitrophenylation after digestion of labeled peptidoglycan with the T7 lysate. Hence the T7 lytic enzyme is an N-acetylmuramyl-l-alanine amidase. It is most likely the product of the T7 lysozyme gene (gene 3.5), since no lytic activity was detected in a lysate prepared by infection of E. coli with a T7 lysozyme mutant.

Keywords

Affiliated Institutions

• State University of New York US
• Stony Brook University US

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