LysK, the enzyme lysing Staphylococcus aureus cells: Specific kinetic features and approaches towards stabilization

Filatova, L. Yu.; Becker, Stephen C.; Donovan, David M.; Gladilin, Alexander K.; Klyachko, Natalia L.

doi:10.1016/j.biochi.2010.01.026

Cited by 41 publications

(28 citation statements)

References 15 publications

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“…2). Whereas most endolysins irreversibly lose their enzymatic activities upon exposure to temperatures around 50°C (37,(39)(40)(41)(42)(43), some thermoresistant endolysins have also been described, including Lys68 from Salmonella phage phi68 (35), gp146 from Salmonella phage PVP-SE1 (21), the endolysins from bacteriophages Ph2119 and vB_Tsc2631 infecting the thermophile Thermus scotoductus (44,45), and the lysin from deep-sea thermophilic bacteriophage GVE2 (46). Gp110 shows an intermediate profile with elevated temperature resistance compared to that of most mesophilic endolysins, which may correlate to increased shelf life of Gp110.…”

Section: Discussionmentioning

confidence: 99%

DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N -Acetylmuramidase Activity

Rodríguez-Rubio

Gerstmans

Thorpe

et al. 2016

Appl Environ Microbiol

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Bacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novel Salmonella bacteriophage endolysin, Gp110, which comprises an uncharacterized domain of unknown function (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/M) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7-to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 has N-acetylmuramidase (lysozyme) activity cleaving the ␤-(1,4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues. Gp110 is active against directly crosslinked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents. IMPORTANCEWe report the functional and biochemical characterization of the Salmonella phage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 has N-acetylmuramidase activity against directly crosslinked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

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Section: Discussionmentioning

confidence: 99%

DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N -Acetylmuramidase Activity

Rodríguez-Rubio

Gerstmans

Thorpe

et al. 2016

Appl Environ Microbiol

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“…We employed commonly used protein stabilizers for expression of soluble proteins in E. coli and obtained counterintuitive results with some of them. Sorbitol is used quite commonly as a stabilizer of proteins in vitro (13,21). It is able to inhibit the unfolding of native conformations to the unfolded/ misfolded forms by a mechanism similar to that of other polyhydric alcohols (20,39).…”

Section: Discussionmentioning

confidence: 99%

Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

Prasad

Khadatare

Roy

2011

Appl Environ Microbiol

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The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium.Overexpression of proteins in heterologous systems often results in the formation of inclusion bodies. These electronrefractile particles are formed because of the failure of prokaryotic folding machinery to correctly fold the nascent polypeptide chain, increasing its local concentration in the cytosol. The most common strategy to recover active proteins from these particles is by denaturing them in the presence of chaotropes and refolding them to the native conformation by the gradual removal of the denaturant. This process results in high loss in protein yield since the renaturation efficiency of most proteins is not very high.

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“…A large number of articles have been published about this phage endolysin (2,6,11) due to its potency and promise as an antibacterial agent.…”

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confidence: 99%

Comparison of the Antibacterial Properties of Phage Endolysins SAL-1 and LysK

Jun

Jung

Son

et al. 2011

Antimicrob Agents Chemother

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In spite of the high degree of amino acid sequence similarity between the newly discovered phage endolysin SAL-1 and the phage endolysin LysK, SAL-1 has an approximately 2-fold-lower MIC against several Staphylococcus aureus strains and higher bacterial cell-wall-hydrolyzing activity than LysK. The amino acid residue change contributing the most to this enhanced enzymatic activity is a change from glutamic acid to glutamine at the 114th residue.Staphylococcus aureus is a highly virulent human pathogen, and S. aureus infections are a significant cause of morbidity and mortality, particularly in settings such as hospitals, nursing homes, and infirmaries (24). In addition to the more severe consequences of contact with S. aureus, this pathogen is also responsible for many cases of food poisoning (14). Many recent isolates of S. aureus show innate resistance to currently available antibiotics (18), leading to challenges in managing S. aureus infections.Since the discovery of bacteriophages, their destructive effect on their host organisms has been exploited as a way of treating infectious bacteria (10). In addition, phage endolysins, also termed lysins, have been proposed as potent antibacterial agents (15,17). Phage endolysins derived from bacteriophages are bacteriophage-encoded peptidoglycan hydrolases that have evolved to rapidly break down the bacterial cell wall, thereby allowing the release of phage progeny (30). Many phage endolysins have shown promise in preclinical trials involving animal models of human diseases (3,5,7,16,20,27), and they thus constitute a promising route for the discovery and development of novel antibacterial therapeutic agents.The phage endolysin LysK from staphylococcal phage K (22) is a valuable endolysin due to its broad-spectrum activity against the staphylococcal genus (23). A large number of articles have been published about this phage endolysin (2, 6, 11) due to its potency and promise as an antibacterial agent.The present study was prompted by the serendipitous observation that phage endolysin SAL-1, derived from the bacteriophage SAP-1 recently isolated in our laboratory, had a significantly lower MIC than phage endolysin LysK in spite of the high level of amino acid sequence similarity between the two proteins. Based on sequencing studies, we determined that phage endolysin SAL-1 only differs from LysK at three residues: isoleucine instead of valine at the 26th residue, glutamine instead of glutamic acid at the 114th residue, and histidine instead of glutamine at the 486th residue.This article describes a comparative study of the newly discovered phage endolysin SAL-1 and the well-studied phage endolysin LysK.An S. aureus bacteriophage was isolated from an environmental sample by a conventional method (29) and designated bacteriophage SAP-1. The gene encoding the endolysin of bacteriophage SAP-1 was identified by comparison with reported sequences and subcloned into the pBAD-TOPO vector (Invitrogen, Carlsbad, CA) by conventional PCR cloning methods. Escherichia coli TOP10 (I...

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LysK, the enzyme lysing Staphylococcus aureus cells: Specific kinetic features and approaches towards stabilization

Cited by 41 publications

References 15 publications

DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N -Acetylmuramidase Activity

DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N -Acetylmuramidase Activity

Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

Comparison of the Antibacterial Properties of Phage Endolysins SAL-1 and LysK

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