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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

Active site of LytM and related proteins: LytM (current structure), Lss—lysostaphin from Staphylococcus simulans (4QPB), LasA from Pseudomonas aeruginosa (3IT5).Orange spheres—zinc ions, red spheres—water molecules coordinating zinc ions.
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f4: Active site of LytM and related proteins: LytM (current structure), Lss—lysostaphin from Staphylococcus simulans (4QPB), LasA from Pseudomonas aeruginosa (3IT5).Orange spheres—zinc ions, red spheres—water molecules coordinating zinc ions.

Mentions: The co-crystal structure of LytM with the phosphinate does not only clarify the role of residues in the active site previously suspected to be involved in catalysis. It also suggests that a residue not previously implicated in LytM catalysis, Tyr204, may make an important contribution as it was suggested for a corresponding residue in LasA7. The hydroxyl group of Tyr204 donates a hydrogen bond to the oxygen atom of the phosphinate that corresponds to the proR oxygen atom of the transition state, which we have argued is derived from the scissile amide bond. In the absence of an experimental structure of LytM with a substrate, we cannot be sure where the carbonyl would be located in the Michaelis complex, but the interpretation of proS and proR oxygens suggests that it would be located between these atoms. If this deduction is correct, then the tyrosine is better positioned to interact with the substrate carbonyl oxygen atom in the transition state than in the ground state. In other words, Tyr204 would be expected to make a significant contribution to catalysis, somewhat akin to the hydrogen bond donors contributing to the oxyanion binding in serine peptidases, which also stabilize the tetrahedral intermediate of the reaction23. Mutational analysis confirmed the significance of Tyr204 for LytM catalysis. We have generated a set of Tyr204 mutants (glycine, alanine, serine, phenylalanine, aspartate and asparagine) and tested them in binding and lytic activity assays. In all tested mutants the activity of the enzyme was very weak or even completely abolished (Fig. 3A) while peptidoglycan binding remained almost unchanged (Fig. 3B). Interestingly, a tyrosine is found in spatially equivalent position also in LasA, even though it is located elsewhere in the amino acid sequence (Fig. 4). LytM and LasA are representative in this respect. In LytM and other closely related enzymes of the M23B subfamily, the tyrosine is always present close to the N-terminal end, within loop 1 (for a nomenclature of loops, see Firczuk et al., 20056 and Spencer et al., 20107). In LasA and other M23A family enzymes, the structurally equivalent tyrosine is located in the C-terminal part of the sequence. Convergent evolution of a tyrosine in proximity to the proR oxygen atom of the transition state further supports a functional role of this tyrosine residue.


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

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

Active site of LytM and related proteins: LytM (current structure), Lss—lysostaphin from Staphylococcus simulans (4QPB), LasA from Pseudomonas aeruginosa (3IT5).Orange spheres—zinc ions, red spheres—water molecules coordinating zinc ions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Active site of LytM and related proteins: LytM (current structure), Lss—lysostaphin from Staphylococcus simulans (4QPB), LasA from Pseudomonas aeruginosa (3IT5).Orange spheres—zinc ions, red spheres—water molecules coordinating zinc ions.
Mentions: The co-crystal structure of LytM with the phosphinate does not only clarify the role of residues in the active site previously suspected to be involved in catalysis. It also suggests that a residue not previously implicated in LytM catalysis, Tyr204, may make an important contribution as it was suggested for a corresponding residue in LasA7. The hydroxyl group of Tyr204 donates a hydrogen bond to the oxygen atom of the phosphinate that corresponds to the proR oxygen atom of the transition state, which we have argued is derived from the scissile amide bond. In the absence of an experimental structure of LytM with a substrate, we cannot be sure where the carbonyl would be located in the Michaelis complex, but the interpretation of proS and proR oxygens suggests that it would be located between these atoms. If this deduction is correct, then the tyrosine is better positioned to interact with the substrate carbonyl oxygen atom in the transition state than in the ground state. In other words, Tyr204 would be expected to make a significant contribution to catalysis, somewhat akin to the hydrogen bond donors contributing to the oxyanion binding in serine peptidases, which also stabilize the tetrahedral intermediate of the reaction23. Mutational analysis confirmed the significance of Tyr204 for LytM catalysis. We have generated a set of Tyr204 mutants (glycine, alanine, serine, phenylalanine, aspartate and asparagine) and tested them in binding and lytic activity assays. In all tested mutants the activity of the enzyme was very weak or even completely abolished (Fig. 3A) while peptidoglycan binding remained almost unchanged (Fig. 3B). Interestingly, a tyrosine is found in spatially equivalent position also in LasA, even though it is located elsewhere in the amino acid sequence (Fig. 4). LytM and LasA are representative in this respect. In LytM and other closely related enzymes of the M23B subfamily, the tyrosine is always present close to the N-terminal end, within loop 1 (for a nomenclature of loops, see Firczuk et al., 20056 and Spencer et al., 20107). In LasA and other M23A family enzymes, the structurally equivalent tyrosine is located in the C-terminal part of the sequence. Convergent evolution of a tyrosine in proximity to the proR oxygen atom of the transition state further supports a functional role of this tyrosine residue.

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