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PMID:22715947

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Citation

Yang, DC, Tan, K, Joachimiak, A and Bernhardt, TG (2012) A conformational switch controls cell wall-remodelling enzymes required for bacterial cell division. Mol. Microbiol. 85:768-81

Abstract

Remodelling of the peptidoglycan (PG) exoskeleton is intimately tied to the growth and division of bacteria. Enzymes that hydrolyse PG are critical for these processes, but their activities must be tightly regulated to prevent the generation of lethal breaches in the PG matrix. Despite their importance, the mechanisms regulating PG hydrolase activity have remained elusive. Here we investigate the control of cell division hydrolases called amidases (AmiA, AmiB and AmiC) required for Escherichia coli cell division. Poorly regulated amiB mutants were isolated encoding lytic AmiB variants with elevated basal PG hydrolase activities in vitro. The structure of an AmiB orthologue was also solved, revealing that the active site of AmiB is occluded by a conserved alpha helix. Strikingly, most of the amino acid substitutions in the lytic AmiB variants mapped to this domain and are predicted to disrupt its interaction with the active site. Our results therefore support a model in which cell separation is stimulated by the reversible relief of amidase autoinhibition governed by conserved subcomplexes within the cytokinetic ring. Analogous conformational control mechanisms are likely to be part of a general strategy used to control PG hydrolases present within multienzyme PG-remodelling machines.

Links

PubMed PMC3418388 Online version:10.1111/j.1365-2958.2012.08138.x

Keywords

Amino Acid Sequence; Cell Division; Cell Wall/enzymology; Cell Wall/metabolism; Crystallography, X-Ray; Escherichia coli/enzymology; Escherichia coli/growth & development; Models, Biological; Molecular Sequence Data; Mutant Proteins/chemistry; Mutant Proteins/genetics; Mutant Proteins/metabolism; Mutation, Missense; N-Acetylmuramoyl-L-alanine Amidase/chemistry; N-Acetylmuramoyl-L-alanine Amidase/genetics; N-Acetylmuramoyl-L-alanine Amidase/metabolism; Protein Conformation; Sequence Alignment

Significance

Annotations

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

BARHE:A0A0H3M3F4

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

ECO:0000315:

F

Figure 1D shows that AmiB mutants have lytic activity. This indicates that these mutants which are increasingly in its active state have increased amidase activity. This increase in unregulated amidase activity results in cell lysis. Figure 2 shows sequence alignment identity and similarities with phage endolysins, lysis enzymes for sporulating bacteria, and lytic enzymes for gram-negative proteobacteria. When compared to phage endolysin there are similar (grey) and identical residues (black) as well as a 50 amino acid section that is not present in phage endolysins that is considered to be used for regulation. The function of this regulation section is described in Figure 3C an alpha helical domain that blocks the substrate from the zinc ion in the active site. Figure 3A and B shows that the core structure of this enzyme is similar to other amidases with exception to the 50 amino acid stretch. Figure S3A (supplemental 3A) shows that three residues that coordinates to the zinc ion in the active site is conserved for LytC-type amidases. Figure 4a shows increased peptidoglycan hydrolase activity for PG sacculi compared to wildtype AmiB when incubated with AmiB mutants that have destabilized regulatory mechanism.

complete
CACAO 12912

Notes

See also

References

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