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Identification and characterization of the metal ion‐dependent l ‐alanoyl‐ d ‐glutamate peptidase encoded by bacteriophage T5
Author(s) -
Mikoulinskaia Galina V.,
Odinokova Irina V.,
Zimin Andrei A.,
Lysanskaya Valentina Ya.,
Feofanov Sergei A.,
Stepnaya Olga A.
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07443.x
Subject(s) - lysin , bacteriophage , lytic cycle , biology , biochemistry , proteus vulgaris , peptidoglycan , escherichia coli , hydrolase , microbiology and biotechnology , enzyme , gene , virus , virology
Although bacteriophage T5 is known to have lytic proteins for cell wall hydrolysis and phage progeny escape, their activities are still unknown. This is the first report on the cloning, expression and biochemical characterization of a bacteriophage T5 lytic hydrolase. The endolysin‐encoding lys gene of virulent coliphage T5 was cloned in Escherichia coli cells, and an electrophoretically homogeneous product of this gene was obtained with a high yield (78% of total activity). The protein purified was shown to be an l ‐alanoyl‐ d ‐glutamate peptidase. The enzyme demonstrated maximal activity in diluted buffers (25–50 m m ) at pH 8.5. The enzyme was strongly inhibited by EDTA and BAPTA, and fully reactivated by calcium/manganese chlorides. It was found that, along with E. coli peptidoglycan, peptidase of bacteriophage T5 can lyse peptidoglycans of other Gram‐negative microorganisms ( Pectobacterium carotovorum , Pseudomonas putida , Proteus vulgaris , and Proteus mirabilis ). This endolysin is the first example of an l ‐alanoyl‐ d ‐glutamate peptidase in a virulent phage infecting Gram‐negative bacteria. There are, however, a great many sequences in databases that are highly similar to that of bacteriophage T5 hydrolase, indicating a wide distribution of endolytic l ‐alanoyl‐ d ‐glutamate peptidases. The article discusses how an enzyme with such substrate specificity could be fixed in the process of evolution.