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Updated: Apr 1, 2026
X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
High resolution structure of an M23 peptidase with a substrate analogue
Maja Grabowska1, Elzbieta Jagielska1, Honorata Czapinska1,2
1International Institute of Molecular and Cell Biology, Ks. Trojdena 4, 02-109 Warsaw, Poland.
Abstract:
LytM is a Staphylococcus aureus autolysin and a homologue of the S. simulans lysostaphin. Both enzymes are members of M23 metallopeptidase family (MEROPS) comprising primarily bacterial peptidoglycan hydrolases. LytM occurs naturally in a latent form, but can be activated by cleavage of an inhibitory N-terminal proregion. Here, we present a 1.45 Å crystal structure of LytM catalytic domain with a transition state analogue, tetraglycine phosphinate, bound in the active site. In the electron density, the active site of the peptidase, the phosphinate and the "diglycine" fragment on the P1' side of the transition state analogue are very well defined. The density is much poorer or even absent for the P1 side of the ligand. The structure is consistent with the involvement of His260 and/or His291 in the activation of the water nucleophile and suggests a possible catalytic role for Tyr204, which we confirmed by mutagenesis. Possible mechanisms of catalysis and the structural basis of substrate specificity are discussed based on the structure analysis.
Insights
The crystal structure of Staphylococcus aureus LytM reveals its catalytic domain and active site. This study elucidates the enzyme's catalytic mechanism and substrate specificity, offering insights into bacterial peptidoglycan hydrolysis.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- LytM is a Staphylococcus aureus autolysin and a metallopeptidase from the M23 family.
- It functions as a bacterial peptidoglycan hydrolase, homologous to S. simulans lysostaphin.
- LytM exists in a latent form, activated by N-terminal proregion cleavage.
Purpose of the Study:
- To determine the crystal structure of the LytM catalytic domain.
- To investigate the enzyme's active site and catalytic mechanism.
- To understand the structural basis of LytM's substrate specificity.
Main Methods:
- X-ray crystallography (1.45 Å resolution).
- Co-crystallization with a transition-state analogue (tetraglycine phosphinate).
- Site-directed mutagenesis (Tyr204).
Main Results:
- The crystal structure reveals the LytM catalytic domain with bound tetraglycine phosphinate.
- The active site, phosphinate, and P1' fragment were well-defined in the electron density.
- His260/His291 involvement in nucleophile activation and Tyr204's catalytic role were suggested and confirmed.
- Poor density on the P1 side suggests flexibility or disorder.
Conclusions:
- The structure provides insights into LytM's catalytic mechanism, involving His residues and Tyr204.
- It lays the groundwork for understanding substrate specificity in M23 metallopeptidases.
- This research contributes to understanding bacterial cell wall hydrolysis and autolysin function.
