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PMID:16103125

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Citation

Low, LY, Yang, C, Perego, M, Osterman, A and Liddington, RC (2005) Structure and lytic activity of a Bacillus anthracis prophage endolysin. J. Biol. Chem. 280:35433-9

Abstract

We report a structural and functional analysis of the lambda prophage Ba02 endolysin (PlyL) encoded by the Bacillus anthracis genome. We show that PlyL comprises two autonomously folded domains, an N-terminal catalytic domain and a C-terminal cell wall-binding domain. We determined the crystal structure of the catalytic domain; its three-dimensional fold is related to that of the cell wall amidase, T7 lysozyme, and contains a conserved zinc coordination site and other components of the catalytic machinery. We demonstrate that PlyL is an N-acetylmuramoyl-L-alanine amidase that cleaves the cell wall of several Bacillus species when applied exogenously. We show, unexpectedly, that the catalytic domain of PlyL cleaves more efficiently than the full-length protein, except in the case of Bacillus cereus, and using GFP-tagged cell wall-binding domain, we detected strong binding of the cell wall-binding domain to B. cereus but not to other species tested. We further show that a related endolysin (Ply21) from the B. cereus phage, TP21, shows a similar pattern of behavior. To explain these data, and the species specificity of PlyL, we propose that the C-terminal domain inhibits the activity of the catalytic domain through intramolecular interactions that are relieved upon binding of the C-terminal domain to the cell wall. Furthermore, our data show that (when applied exogenously) targeting of the enzyme to the cell wall is not a prerequisite of its lytic activity, which is inherently high. These results may have broad implications for the design of endolysins as therapeutic agents.

Links

PubMed Online version:10.1074/jbc.M502723200

Keywords

Amidohydrolases/chemistry; Amidohydrolases/physiology; Amino Acid Sequence; Bacillus anthracis/metabolism; Bacterial Proteins; Binding Sites; Catalytic Domain; Cell Wall/metabolism; Cloning, Molecular; Endopeptidases/chemistry; Genome, Bacterial; Models, Biological; Models, Molecular; Models, Statistical; Molecular Sequence Data; N-Acetylmuramoyl-L-alanine Amidase/chemistry; Peptidoglycan/chemistry; Prophages/chemistry; Prophages/metabolism; Protein Binding; Protein Conformation; Protein Folding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Species Specificity; Time Factors; Zinc/chemistry

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

9CAUD:Q8LTE6

Contributes to

GO:0008745: N-acetylmuramoyl-L-alanine amidase activity

ECO:0000247: sequence alignment evidence used in manual assertion

UniProtKB:Q81WA9


F

Zinc binding and catalysis is highly conserved between Bacillus phages’ endolysins. This is indicated by the blue and red triangles located under the specific residues in the Figure 1. There is a high sequence alignment between PlyG and PlyLBa02 as shown by Figure 1 which is a sequence alignment diagram from ClustalX. PlyLBA02 and PlyG have a 93% identity in the enzymatic domain and a 60% identity in the C-terminal domain. Because of this similarity and the conserved residues, we can come to conclusion the PlyG is also involved in N-acetylmuramoyl-L-alanine amidase activity.

complete
CACAO 12920

BACAN:Q81WA9

GO:0008745: N-acetylmuramoyl-L-alanine amidase activity

ECO:0000314:

F

PlyL is an endolysin enzyme with 2 separate domains, an N-terminal catalytic domain, and a C-terminal cell wall binding domain. This annotation is for the N-terminal domain of PlyL.

Peptidoglycan from B. subtilis was subjected to both the full-length PlyL and the N-terminal domain only. In supplemental figure 1, (see link: http://www.jbc.org/content/suppl/2005/08/16/M502723200.DC1/Suppdata.pdf) the amount of 2,4-dinitrophenyl-alanine is shown to increase sharply, indicating that PlyL is an N-acetylmuramoyl-L-alanine amidase. Additionally, the figure shows that alanine is increased even more when the N-terminal domain alone is used, compared to the full-length protein.

As additional support that PlyL is an amidase, figure 2 compares the crystal structure of PlyL to other known amidase enzymes.

Figure 4 shows a time course assay where the purified endolysin (PlyL) was able to lyse 4 different bacillus species cultures within 400 seconds. In 4a, the lysing capabilities of the full-length version of PlyL are shown. In 4b, the lysing capabilities of the N-terminal portion only are shown. The N-terminal domain by itself is more effective at cleaving Bacillus species than the entirety of PlyL.

complete
CACAO 11527

BACAN:Q81WA9

GO:0051672: catabolism by organism of cell wall peptidoglycan in other organism

ECO:0000314:

P

Peptidoglycan from B. subtilis was subjected to both the full-length PlyL and the N-terminal domain only. In supplemental figure 1, (see link: http://www.jbc.org/content/suppl/2005/08/16/M502723200.DC1/Suppdata.pdf) the amount of 2,4-dinitrophenyl-alanine is shown to increase sharply, indicating that PlyL is an N-acetylmuramoyl-L-alanine amidase. Additionally, the figure shows that alanine is increased even more when the N-terminal domain alone is used, compared to the full-length protein.

complete
CACAO 11533

BACAN:Q81WA9

Contributes to

GO:0008745: N-acetylmuramoyl-L-alanine amidase activity

ECO:0000247:

UniProtKB:Q8LTE6


F

Zinc binding and catalysis is highly conserved between Bacillus phages. There is a high sequence alignment between PlyLBa02 and PlyG as shown by Figure 1 which is a sequence alignment diagram from ClustalX. PlyG and PlyLBA02 have a 93% identity in the enzymatic domain and a 60% identity in the C-terminal domain. Because of this similarity, we can come to conclusion the PlylBa02 is also involved in N-acetylmuramoyl-L-alanine amidase activity.

complete
CACAO 12461

BACAN:Q81WA9

Contributes to

GO:0019835: cytolysis

ECO:0000315:

P

In Figure 4, an assay of lytic enzyme activity of PlyL and its mutant, Ply21 was conducted on the cell wall of five bacterial hosts. The mutant Ply21 is described as the full-length protein of PlyL with the removal of the C-terminal domain involved in cell wall binding, leaving behind only the presence of the N-terminal catalytic domain. In Panel B, the lysing abilities of Ply21 are shown, and the removal of the C-terminal cell wall binding domain did not have a significant effect on the lysing of the bacterial hosts. However, the N-terminal catalytic domain displayed a higher percent absorbance over time as compared to Panel A, (which displayed the lysing abilities of the full length protein). Furthermore, B. subtilis (yellow-orange line) displayed the highest lysing ability than all of the bacterial hosts, with the absorbance rate being higher in the graph of Panel B than Panel A. This concludes that the mutant PlyL 21, also known as the N-terminal catalytic domain of the full length PlyL protein with the C-terminal binding domain removed, is most efficient in cytolysis and that the removal of the C-terminal cell wall binding domain does not inhibit nor improve the lysing ability of PlyL on the cell wall of bacterial hosts.

complete
CACAO 12907

BACAN:Q81WA9

Contributes to

GO:0008745: N-acetylmuramoyl-L-alanine amidase activity

ECO:0000255:

UniProtKB:0008745


F

Figure 5 shows that catalytic activity of N-terminal domain binding to the C-terminal domain in which it shows the C-terminal domain to catalyze the cell wall component. This proves that the N-acetylmuramoyl-L-alanine amidase activity is being used.

complete
CACAO 12910

BACTU:J3UKZ5

GO:0019835: cytolysis

ECO:0000314:

P

Fig. 4A-D demonstrates that the protein has lytic activity by observing percent absorption or time (second) and that in some species the catalytic domain alone yields higher activity than the full length protein while for other species it is the opposite.

complete
CACAO 12550

Notes

See also

References

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