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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

Proposed catalytic mechanism of the LytM class of enzymes.The top left and right bottom panels are based on the actual crystal structures of LasA (3IT5) and LytM (presented here) except that in the complex with the phosphinate His260 is not protonated, and binds the proS oxygen atom instead. The other two panels are models and therefore only the most convincing interactions have been shown but it is possible that Tyr204, His260 and His291 would contribute to the substrate/product binding as well. The contact between the water coordinating Zn2+ ion and the Tyr204 is observed in some structures (3IT5) but not in others (4QPB), and must be weak so that the substrate can displace the water molecule. The LytM numbering has been used in all panels.
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f2: Proposed catalytic mechanism of the LytM class of enzymes.The top left and right bottom panels are based on the actual crystal structures of LasA (3IT5) and LytM (presented here) except that in the complex with the phosphinate His260 is not protonated, and binds the proS oxygen atom instead. The other two panels are models and therefore only the most convincing interactions have been shown but it is possible that Tyr204, His260 and His291 would contribute to the substrate/product binding as well. The contact between the water coordinating Zn2+ ion and the Tyr204 is observed in some structures (3IT5) but not in others (4QPB), and must be weak so that the substrate can displace the water molecule. The LytM numbering has been used in all panels.

Mentions: The tetraglycine phosphinate mimics the transition state of pentaglycine crossbridge hydrolysis. We use proR and proS labels for the oxygen atoms according to the IUPAC rules applied to the transition state intermediate (and not the phosphinate itself) to make the terminology independent of the analogue used to elucidate mechanism. In this convention, the proR and proS oxygen atoms are 1.9 and 3.0 Å away from the Zn2+ cation, respectively (Fig. 1C). The observed distances suggest identification of the transition state proR oxygen atom with the carbonyl oxygen atom of the substrate and of the proS oxygen atom with the incoming water molecule or hydroxide ion. This implies polarization of the scissile amide bond due to the coordination of the carbonyl oxygen atom by the Zn2+ ion in the active site, as in almost all detailed proposals for catalysis by single cation metallopeptidases1718. It also implies that the nucleophilic water molecule or hydroxide ion has to approach from the si face of the amide bond (Fig. 2). In this scenario, the metal ion is not involved in the activation of the nucleophile, as proposed for some but not all metallopeptidases1718. Instead, the structure suggests that the incoming water molecule/hydroxide ion is activated by His260 and His291, as tentatively already proposed in the case of LytM19, and for the equivalent histidines also in the case of the LytM orthologue LasA7. In the experimental structure of the phosphinate complex, the oxygen atom that would correspond to the proS oxygen of the transition state is 2.6 and 2.8 Å away from the Nε atoms of His260 and His291, respectively (Fig. 1C). Loss of activity when either histidine is replaced by alanine in LytM519 (or its lysostaphin20, LasA21 and Ale-122 homologues) supports a role of the histidines in catalysis.


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

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

Proposed catalytic mechanism of the LytM class of enzymes.The top left and right bottom panels are based on the actual crystal structures of LasA (3IT5) and LytM (presented here) except that in the complex with the phosphinate His260 is not protonated, and binds the proS oxygen atom instead. The other two panels are models and therefore only the most convincing interactions have been shown but it is possible that Tyr204, His260 and His291 would contribute to the substrate/product binding as well. The contact between the water coordinating Zn2+ ion and the Tyr204 is observed in some structures (3IT5) but not in others (4QPB), and must be weak so that the substrate can displace the water molecule. The LytM numbering has been used in all panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Proposed catalytic mechanism of the LytM class of enzymes.The top left and right bottom panels are based on the actual crystal structures of LasA (3IT5) and LytM (presented here) except that in the complex with the phosphinate His260 is not protonated, and binds the proS oxygen atom instead. The other two panels are models and therefore only the most convincing interactions have been shown but it is possible that Tyr204, His260 and His291 would contribute to the substrate/product binding as well. The contact between the water coordinating Zn2+ ion and the Tyr204 is observed in some structures (3IT5) but not in others (4QPB), and must be weak so that the substrate can displace the water molecule. The LytM numbering has been used in all panels.
Mentions: The tetraglycine phosphinate mimics the transition state of pentaglycine crossbridge hydrolysis. We use proR and proS labels for the oxygen atoms according to the IUPAC rules applied to the transition state intermediate (and not the phosphinate itself) to make the terminology independent of the analogue used to elucidate mechanism. In this convention, the proR and proS oxygen atoms are 1.9 and 3.0 Å away from the Zn2+ cation, respectively (Fig. 1C). The observed distances suggest identification of the transition state proR oxygen atom with the carbonyl oxygen atom of the substrate and of the proS oxygen atom with the incoming water molecule or hydroxide ion. This implies polarization of the scissile amide bond due to the coordination of the carbonyl oxygen atom by the Zn2+ ion in the active site, as in almost all detailed proposals for catalysis by single cation metallopeptidases1718. It also implies that the nucleophilic water molecule or hydroxide ion has to approach from the si face of the amide bond (Fig. 2). In this scenario, the metal ion is not involved in the activation of the nucleophile, as proposed for some but not all metallopeptidases1718. Instead, the structure suggests that the incoming water molecule/hydroxide ion is activated by His260 and His291, as tentatively already proposed in the case of LytM19, and for the equivalent histidines also in the case of the LytM orthologue LasA7. In the experimental structure of the phosphinate complex, the oxygen atom that would correspond to the proS oxygen of the transition state is 2.6 and 2.8 Å away from the Nε atoms of His260 and His291, respectively (Fig. 1C). Loss of activity when either histidine is replaced by alanine in LytM519 (or its lysostaphin20, LasA21 and Ale-122 homologues) supports a role of the histidines in catalysis.

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