PMID:21816821

Citation	Low, LY, Yang, C, Perego, M, Osterman, A and Liddington, R (2011) Role of net charge on catalytic domain and influence of cell wall binding domain on bactericidal activity, specificity, and host range of phage lysins. J. Biol. Chem. 286:34391-403
Abstract	The recombinant lysins of lytic phages, when applied externally to Gram-positive bacteria, can be efficient bactericidal agents, typically retaining high specificity. Their development as novel antibacterial agents offers many potential advantages over conventional antibiotics. Protein engineering could exploit this potential further by generating novel lysins fit for distinct target populations and environments. However, access to the peptidoglycan layer is controlled by a variety of secondary cell wall polymers, chemical modifications, and (in some cases) S-layers and capsules. Classical lysins require a cell wall-binding domain (CBD) that targets the catalytic domain to the peptidoglycan layer via binding to a secondary cell wall polymer component. The cell walls of Gram-positive bacteria generally have a negative charge, and we noticed a correlation between (positive) charge on the catalytic domain and bacteriolytic activity in the absence of the CBD (nonclassical behavior). We investigated a physical basis for this correlation by comparing the structures and activities of pairs of lysins where the lytic activity of one of each pair was CBD-independent. We found that by engineering a reversal of sign of the net charge of the catalytic domain, we could either eliminate or create CBD dependence. We also provide evidence that the S-layer of Bacillus anthracis acts as a molecular sieve that is chiefly size-dependent, favoring catalytic domains over full-length lysins. Our work suggests a number of facile approaches for fine-tuning lysin activity, either to enhance or reduce specificity/host range and/or bactericidal potential, as required.
Links	PubMed PMC3190764 Online version:10.1074/jbc.M111.244160
Keywords	Bacillus anthracis/genetics; Bacillus anthracis/metabolism; Bacillus anthracis/virology; Bacillus subtilis/genetics; Bacillus subtilis/metabolism; Bacillus subtilis/virology; Bacteriophages/enzymology; Bacteriophages/genetics; Cell Wall/genetics; Cell Wall/metabolism; Cell Wall/virology; Host Specificity/physiology; Hydrolases/genetics; Hydrolases/metabolism; Protein Binding; Protein Engineering; Protein Structure, Tertiary; Viral Proteins/genetics; Viral Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BACAN:Q81YZ2		GO:0003796: lysozyme activity	ECO:0000247:	PMID:3190764	F		Figure 3B shows the sequence of the XlyA catalytic domain is similar to the catalytic domain of PlyL, another endolysin. The red boxes show XlyA contains catalytic and substrate recognition residues similar or identical to PlyL.The asterisks indicate base pair identity between XlyA and PlyL sequences. The colons indicate base pair similarity between XylA and PlyL. The high similarity and identity within the sequence alignment and secondary structure assignments of XylA and PlyL support the lysozyme function of XylA.	complete CACAO 12603
BACAN:Q81YZ2		GO:0003796: lysozyme activity	ECO:0000250:	UniProtKB:Q81YZ2	F		Figure 2B shows the structural homolog of XlyA. XlyA is an endolysin. Figure 2B shows structural similarity between XlyA and another amidase lysin, PlyL.	complete CACAO 12636
BACAN:Q81YZ2		GO:0003796: lysozyme activity	ECO:0000250:		F		In Figure 2A, both XylA and PlyL’s structures were being compared which indicated that the two of these proteins are amidase lysins. In this particular figure(2A), their functions were being tested based on their ability to lyse B. Subtilis. They found that the catalytic domain of PlyL which was indicated in the paper by “PlyL CAT” had homologous lysing abilities to the entirety of XylA.	Missing: with/from CACAO 12646
BACAN:Q81YZ2		GO:0005975: carbohydrate metabolic process	ECO:0000314:		P		Figure 1 shows the use of carboxyl groups on MurNac moiety to for an amide bond with α-amino group of l-Ala. The chemical reaction's use of the carboxyl groups to form additional bonds between peptides.	complete CACAO 12662
BACAN:Q81YZ2		GO:0016998: cell wall macromolecule catabolic process	ECO:0000314:		P		the Effect of S-layer—Was compared the effects of PlyL and they were closely related PlyG with 96% identical in the catalytic domain , and 63% in the CBD. Figure 5A: shows how the cell killing activity of the full length which is the top bar and the catalytic domain the second bar of plyL and plyG against B. anthracis Sterne, B. anthracis (sap) mutant deficient in S-layer synthesis,and B. subtilis. Each B strains consist of 0.4uM of lysin concentration. From the graph we can also conclude that PlyG(CAT) killed Sterne more than twice as fast as PlyG(FULL). The full length graph show that with out S-layer, PlyL and plyG can still be active. The blue bar is plyL (full) and red bar is plyL(CAT). the light blue bar is plyG (full) and green bar is plyG(CAT). PlyL(CAT) had a higher lytic activity than PlyL(FULL), and PlyG(CAT) killed Sterne more than twice as fast as PlyG (FULL)	complete CACAO 12887
BACAN:Q81YZ2		GO:0003796: lysozyme activity	ECO:0000314:		F		Figure 1 displays that the two glycan chains are cross-peptide linked. The bond formed is what the amidase lysins target . It displays the cleavage positions of other common lysins in comparison to this lysin. Through this figure one can see that the gram-positive bacteria displays species as well as strain specific "secondary cell wall polymers" (SCWPS). These insert themselves into the lipid bilayer, which changes the appearance and the charge of the outer envelope. Figure 2 was used in order to compare this lysin to two homologous amidase lysins to see the similar structures and distinct behaviours between them (PlyL and XlyA). Due to the high structural similarity, a test was conducted to see whether the innate catalytic acitivty of XlyA was similar to that of PlyL. Figure 3 displays that XlyA:plyL chimera failed to lyse B. subtilis, however it did lyse B. cereus.	complete CACAO 12909
BACAN:Q81YZ2		GO:0016787: hydrolase activity	ECO:0000314:		F		In Figure 1, SCWPs are typically attached via the C-6 hydroxyl group on MurNac. The presence of the hydroxyl group is indication of hydrolase activity. The d-Glu uses its γ-carboxyl to makes an amide bond with m-DAP. The presence of a carboxyl group, which contains bonds used in hydrolase activity. The γ-carboxyl is used similarly to an enzyme in the process of making an amide bond.	complete CACAO 12918
BACSU:XLYA	Contributes to	GO:0019835: cytolysis	ECO:0000250:	UniProtKB:Q81WA9	P		In Figure 2, the structures and desirable behaviors were compared between PlyL and XlyA, concluding them to be homologous amidase lysins. Specifically in Panel A of Figure 2, the lysing abilities of the strains were compared on their effect on host cell B. Subtilis. The full-length protein of XlyA was similar to its lysing abilities of the catalytic domain of PlyL, which is responsible for producing amidase, an enzyme that cleaves peptidoglycan in the cell walls of the bacterial host cells.	complete CACAO 12917

References

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