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High resolution structure of an M23 peptidase with a substrate analogue.

Grabowska M, Jagielska E, Czapinska H, Bochtler M, Sabala I - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: International 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.

No MeSH data available.


Related in: MedlinePlus

Transition state analogue and inhibitory proregion in the LytM substrate binding groove.(A) Tetraglycine phosphinate and (B) proregion (PDB 1QWY).
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f6: Transition state analogue and inhibitory proregion in the LytM substrate binding groove.(A) Tetraglycine phosphinate and (B) proregion (PDB 1QWY).

Mentions: LytM is naturally made as a latent enzyme with an inhibitory region at its N-terminus, which can be cleaved off even by physiologically unrelated peptidases5. Although such LytM activation in physiological circumstances has never been shown, this region nevertheless behaves biochemically much like a proregion and will be called so in the following. In the previously determined structure of full length LytM5 the proregion runs through the active site cleft, but is not cleaved (Fig. 5B). This can be attributed to a relatively large distance of the peptide backbone from the active site Zn2+ ion. Additionally the Zn2+ ion is trapped in a tetrahedral coordination arrangement with the asparagine carboxamide oxygen atom as the fourth ligand in addition to the three usual ones (His210, Asp214, His293). The current structure makes it possible to relate the proregion and substrate (analogue) binding modes. We found a surprising similarity between the P1′ side of the phosphinate and the 117-NND—119 fragment of the proregion (Fig. 6). Both run in the same direction with the distance between corresponding Cα-atoms below 2.0 Å. The agreement does not hold for the phosphinate group (corresponding to the scissile bond). Whereas the phosphinate runs straight through the active site, the proregion kinks sharply. This kink provides sufficient distance for the “asparagine switch” Asn117 residue to contact the metal via the carbonyl oxygen atom of the side and not main chain. We conclude from this analysis that the proregion of LytM mimics a substrate in the primed, but not in the non-primed region of the substrate binding cleft.


High resolution structure of an M23 peptidase with a substrate analogue.

Grabowska M, Jagielska E, Czapinska H, Bochtler M, Sabala I - Sci Rep (2015)

Transition state analogue and inhibitory proregion in the LytM substrate binding groove.(A) Tetraglycine phosphinate and (B) proregion (PDB 1QWY).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4594094&req=5

f6: Transition state analogue and inhibitory proregion in the LytM substrate binding groove.(A) Tetraglycine phosphinate and (B) proregion (PDB 1QWY).
Mentions: LytM is naturally made as a latent enzyme with an inhibitory region at its N-terminus, which can be cleaved off even by physiologically unrelated peptidases5. Although such LytM activation in physiological circumstances has never been shown, this region nevertheless behaves biochemically much like a proregion and will be called so in the following. In the previously determined structure of full length LytM5 the proregion runs through the active site cleft, but is not cleaved (Fig. 5B). This can be attributed to a relatively large distance of the peptide backbone from the active site Zn2+ ion. Additionally the Zn2+ ion is trapped in a tetrahedral coordination arrangement with the asparagine carboxamide oxygen atom as the fourth ligand in addition to the three usual ones (His210, Asp214, His293). The current structure makes it possible to relate the proregion and substrate (analogue) binding modes. We found a surprising similarity between the P1′ side of the phosphinate and the 117-NND—119 fragment of the proregion (Fig. 6). Both run in the same direction with the distance between corresponding Cα-atoms below 2.0 Å. The agreement does not hold for the phosphinate group (corresponding to the scissile bond). Whereas the phosphinate runs straight through the active site, the proregion kinks sharply. This kink provides sufficient distance for the “asparagine switch” Asn117 residue to contact the metal via the carbonyl oxygen atom of the side and not main chain. We conclude from this analysis that the proregion of LytM mimics a substrate in the primed, but not in the non-primed region of the substrate binding cleft.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: International 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.

No MeSH data available.


Related in: MedlinePlus