Identification and characterization of the metal ion‐dependent l‐alanoyl‐d‐glutamate peptidase encoded by bacteriophage T5

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.

Listeria bacteriophage peptidoglycan hydrolases feature high thermoresistance and reveal increased activity after divalent metal cation substitution

“…Considering that Zn 2+ obviously assumes a crucial role in catalytic function of these enzymes, it was surprising to find that Ca 2+ and Mn 2+ were more effective in restoring lytic activity after . Similar observations regarding the effects of divalent metals on activity of cell wall hydrolases were reported earlier (Vasala et al 1995;Shida et al 2001;Pritchard et al 2004;Donovan et al 2006;Sugahara et al 2007;Mikoulinskaia et al 2009;Garcia et al 2010). For aminopeptidase A, a zinc metalloendopeptidase that can be reactivated by Ca 2+ or Mn 2+ after exposure to chelating agents (Danielsen et al 1980), it was recently shown that the Ca 2+ ion increases substrate affinity by binding to two aspartate residues of the enzyme and interacting with an acidic side chain of the substrate (Claperon et al 2008).…”

“…In fact, most other phage and bacterial lysins studied require neutral or slightly acidic conditions, with activity rapidly decreasing above pH 7.5 or 8 (Vasala et al 1995;Morita et al 2001;Loeffler et al 2003;Pritchard et al 2004;Yoong et al 2004;Cheng and Fischetti 2006;Donovan et al 2006;Fukushima et al 2007;Sugahara et al 2007). However, another L-alanoyl-D-glutamate peptidase from E. coli phage T5 revealed a pH profile similar to the enzymes reported here, with highest activity at pH 8.5 (Mikoulinskaia et al 2009). It is interesting to note that these properties correspond well to the optimum range for Listeria, which also grows best at a pH of around 8.0 (Vanderzant and Splittstoesser 1992).…”

“…Two different enzymatic specificities of Listeria phage endolysins have been reported: N-acetylmuramoyl-L-alanine amidases and L-alanoyl-D-glutamate peptidases. The latter was first identified in Ply118 and Ply500 (Loessner et al 1995b) and has recently also been reported for a Bacillus subtilis enzyme (Fukushima et al 2007) and Escherichia coli phage T5 endolysin (Mikoulinskaia et al 2009). Like most known endolysins, the Listeria phage enzymes show a modular organization: an N-terminal enzymatically active domain (EAD) is linked to a Cterminal cell wall binding domain (CBD), which directs the enzyme to a cell wall-associated ligand (Korndoerfer et al 2006;Loessner et al 2002;Schmelcher et al 2010).…”

Molecular Aspects and Comparative Genomics of Bacteriophage Endolysins

“…In opposition to what is described by Pfam, the Listeria phage endolysins Ply118 and Ply500 and the enterobacteriophage T5 endolysin classified as VANY carboxypeptidases act as L-alanyl-D-glutamate endopeptidases (56,57). PET-M15-4 is a domain also described as D-alanyl-D-alanine carboxypeptidase; however, it has been predicted to display L-Ala-D-Glu activity (54).…”

Genome analysis of Cronobacter phage vB_CsaP_Ss1 reveals an endolysin with potential for biocontrol of Gram-negative bacterial pathogens

“…Conserved domain analysis revealed the presence of a VanY Pfam (SMART) and a peptidase M15_4 (CDD), both of which are described as a D-alanyl-D-alanine carboxypeptidase. Contrary to what has been described, it has been experimentally reported that Listeria phage endolysins Ply118 and Ply500 and the endolysin from bacteriophage T5, although classified as putative D-alanyl-D-alanine carboxypeptidases, actually display L-alanyl-D-glutamate activity (Loessner et al, 1995;Mikoulinskaia et al, 2009;Payne & Hatfull, 2012 Cerebrospinal fluid…”