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

Abstract

The ability of the bacteriophage-encoded peptidoglycan hydrolases (endolysins) to destroy Gram-positive bacteria from without makes these enzymes promising antimicrobials. Recombinant endolysins from Listeria monocytogenes phages have been shown to rapidly lyse and kill the pathogen in all environments. To determine optimum conditions regarding application of recombinant Listeria phage endolysins in food or production equipments, properties of different Listeria endolysins were studied. Optimum NaCl concentration for the amidase HPL511 was 200 nM and 300 mM for the peptidases HPL118, HPL500, and HPLP35. Unlike most other peptidoglycan hydrolases, all four enzymes exhibited highest activity at elevated pH values at around pH 8-9. Lytic activity was abolished by EDTA and could be restored by supplementation with various divalent metal cations, indicating their role in catalytic function. While substitution of the native Zn 2+ by Ca 2+ or Mn 2+ was most effective in case of HPL118, HPL500, and HPLP35, supplementation with Co 2+ and Mn 2+ resulted in an approximately 5-fold increase in HPL511 activity. Interestingly, the glutamate peptidases feature a conserved SxHxxGxAxD zinc-binding motif, which is not present in the amidases, although they also require centrally located divalent metals for activity.The endolysins HPL118, HPL511, and HPLP35 revealed a surprisingly high thermostability, with up to 35% activity remaining after 30 min incubation at 90°C. The available data suggest that denaturation at elevated temperatures is reversible and may be followed by rapid refolding into a functional state.

Lytic Activity of the Vibrio cholerae Type VI Secretion Toxin VgrG-3 Is Inhibited by the Antitoxin TsaB

“…Incubation with the cation scavenger EDTA did not diminish activity significantly, indicating that the cation cofactor is not essential but may play an accessory role by assisting in folding or stabilization of the active site of the enzyme (17). -3N), and its C-terminal extension (VgrG-3C) (Fig.…”

“…In this study, the purified recombinant C-terminal extension of VgrG-3 (VgrG-3C) was run in each lane of a zymogram gel and renatured in buffers at different pHs (5.0 -9.0) or at pH 7 in the presence of various cations as the activity of many peptidoglycan hydrolases has been shown to be dependent on cation cofactors (17,18). The results of these analyses (Fig.…”

Roles of bacteriophage GVE2 endolysin in host lysis at high temperatures

“…Several endolysins from mesophilic bacteriophages have been reported to possess the feature of extreme thermoresistance (up to 90 u C) (Walmagh et al, 2012). The thermoresistance of this group of endolysins may be due to rapid refolding of endolysins upon cooling, as suggested by Schmelcher and coworkers (Schmelcher et al, 2012). But no conserved motif was found between GVE2 endolysin and thermostable endolysin from mesophilic bacteriophages according to BLAST analysis.…”

Cloning and Characterization of a Novel N-acetylglucosaminidase (AtlD) from Enterococcus faecalis

“…It retained 85% of the relative activity with NaCl 1,000 mM, which means that in the salt concentration found in many food products (200-300 mM), such as fermented meat products and cheeses (until 1 M in Cotija cheese), the enzyme could show its optimal activity. This could represent an advantage over other PGH, which act better in physiological conditions (130 mM) or even in the absence of NaCl [Fukushima et al, 2007;Schmelcher et al, 2012].…”