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PMID:19919545

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Contents

Citation

Mikoulinskaia, GV, Odinokova, IV, Zimin, AA, Lysanskaya, VY, Feofanov, SA and Stepnaya, OA (2009) Identification and characterization of the metal ion-dependent L-alanoyl-D-glutamate peptidase encoded by bacteriophage T5. FEBS J. 276:7329-42

Abstract

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 mM) 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.

Links

PubMed Online version:10.1111/j.1742-4658.2009.07443.x

Keywords

Amino Acid Sequence; Bacteriolysis; Calcium Chloride/pharmacology; Chlorides/pharmacology; Cloning, Molecular; Edetic Acid/pharmacology; Egtazic Acid/analogs & derivatives; Egtazic Acid/pharmacology; Endopeptidases/isolation & purification; Endopeptidases/metabolism; Manganese Compounds/pharmacology; Microbial Viability/drug effects; Molecular Sequence Data; Peptidoglycan/metabolism; Protease Inhibitors/pharmacology; Sequence Alignment; Siphoviridae/enzymology; Substrate Specificity; Viral Proteins/isolation & purification; Viral Proteins/metabolism

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Annotations

Gene product Qualifier GO ID GO term name Evidence Code with/from Aspect Notes Status

Phage_T5_lys:Gene_Product(s)

GO:0051672

catabolism by organism of cell wall peptidoglycan in other organism

IDA: Inferred from Direct Assay

P

complete

Phage_T5_lys:Gene_Product(s)

GO:0004222

metalloendopeptidase activity

IDA: Inferred from Direct Assay

F

complete

Phage_T5_lys:Gene_Product(s)

GO:0046872

metal ion binding

IDA: Inferred from Direct Assay

F

complete

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