insight - Biochemistry - # Substrate specificity of peptidoglycan hydrolases in Staphylococcus aureus

Deciphering the Substrate Specificities of the Major Staphylococcus aureus Peptidoglycan Hydrolases Lysostaphin and LytM

Q: How do the substrate specificities of LSS and LytM influence their potential as antimicrobial agents against S. aureus, particularly against strains with modified peptidoglycan compositions?

The substrate specificities of LSS and LytM play a crucial role in determining their effectiveness as antimicrobial agents against S. aureus, especially in the context of strains with modified peptidoglycan compositions. LSS, being a glycyl-glycine endopeptidase, shows a preference for cleaving the glycyl-glycine bonds in the pentaglycine cross-bridge of S. aureus peptidoglycan. This specificity makes LSS highly effective against S. aureus strains with the typical pentaglycine cross-bridge structure in their peptidoglycan. On the other hand, LytM, in addition to its glycyl-glycine endopeptidase activity, has been shown to exhibit D-Ala-Gly endopeptidase activity. This expanded substrate specificity allows LytM to target the D-Ala-Gly cross-links in the peptidoglycan of S. aureus. As a result, LytM may be more effective against S. aureus strains with modified peptidoglycan compositions, such as those with altered cross-bridge structures or substitutions like serine in the cross-bridge. The ability of LytM to target different types of bonds in the peptidoglycan provides it with a broader spectrum of antimicrobial activity compared to LSS. In summary, the substrate specificities of LSS and LytM influence their antimicrobial potential by determining the types of bonds they can cleave in the peptidoglycan structure of S. aureus. This specificity allows them to target specific components of the bacterial cell wall, making them effective antimicrobial agents against different strains of S. aureus with varying peptidoglycan compositions.

Core Concepts

The major Staphylococcus aureus peptidoglycan hydrolases lysostaphin and LytM display distinct substrate specificities, with lysostaphin preferring the D-Ala-Gly cross-link in mature peptidoglycan, while LytM can also cleave the D-alanyl-glycine bond in addition to glycyl-glycine bonds.

Abstract

The content provides a detailed analysis of the substrate specificities of the major Staphylococcus aureus peptidoglycan hydrolases, lysostaphin and LytM, using NMR spectroscopy and turbidity reduction assays.

The key findings are:

Lysostaphin (LSS) is a glycyl-glycine endopeptidase that strongly prefers substrates containing the D-Ala-Gly cross-link, which increases its catalytic efficiency 10-fold compared to non-cross-linked substrates. LSS primarily cleaves the bond between Gly1 and Gly2 in the pentaglycine cross-bridge.
Contrary to previous understanding, the S. aureus peptidoglycan hydrolase LytM is not only a glycyl-glycine endopeptidase, but can also cleave the D-alanyl-glycine bond in addition to glycyl-glycine bonds. LytM displays flexibility in accommodating either D-Ala or Gly at the P1 position.
The structural differences between LSS and LytM, particularly in the loop regions surrounding the active site, explain their distinct substrate specificities. LSS has a longer loop that sterically hinders the binding of D-Ala, while the shorter loop in LytM allows it to accommodate D-Ala in the active site.
The substrate specificities of LSS and LytM provide a structural-level understanding of their functional roles and help explain the observed differences in lytic efficiencies against S. aureus strains with varying peptidoglycan compositions.

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biorxiv.org

Stats

The pentaglycine cross-bridge in S. aureus peptidoglycan is composed of five glycine residues.
Lysostaphin hydrolyzes the bond between Gly1 and Gly2 in the pentaglycine cross-bridge at a rate 15-fold faster than LytM.
Lysostaphin hydrolyzes the D-Ala-Gly cross-link 10-fold faster than non-cross-linked substrates.
LytM can hydrolyze both the D-Ala-Gly and Gly1-Gly2 bonds in the pentaglycine cross-bridge.

Quotes

"LSS prefers cutting between Gly1 and Gly2, whenever Gly1 is cross-linked to D-Ala of neighboring stem, whereas it hydrolyses the amide bond between Gly2 and Gly3 in non-cross-linked (devoid of D-Ala-Gly bond) PG fragments."
"Contrary to previous understanding, the S. aureus peptidoglycan hydrolase LytM is not only a glycyl-glycine endopeptidase, but can also cleave the D-alanyl-glycine bond in addition to glycyl-glycine bonds."

Key Insights Distilled From

Reassessing the substrate specificities of the major Staphylococcus aureus peptidoglycan hydrolases lysostaphin and LytM

by Ante... at www.biorxiv.org 10-13-2023

https://www.biorxiv.org/content/10.1101/2023.10.13.562287v2

Deeper Inquiries

How do the substrate specificities of LSS and LytM influence their potential as antimicrobial agents against S. aureus, particularly against strains with modified peptidoglycan compositions?

The substrate specificities of LSS and LytM play a crucial role in determining their effectiveness as antimicrobial agents against S. aureus, especially in the context of strains with modified peptidoglycan compositions. LSS, being a glycyl-glycine endopeptidase, shows a preference for cleaving the glycyl-glycine bonds in the pentaglycine cross-bridge of S. aureus peptidoglycan. This specificity makes LSS highly effective against S. aureus strains with the typical pentaglycine cross-bridge structure in their peptidoglycan.
On the other hand, LytM, in addition to its glycyl-glycine endopeptidase activity, has been shown to exhibit D-Ala-Gly endopeptidase activity. This expanded substrate specificity allows LytM to target the D-Ala-Gly cross-links in the peptidoglycan of S. aureus. As a result, LytM may be more effective against S. aureus strains with modified peptidoglycan compositions, such as those with altered cross-bridge structures or substitutions like serine in the cross-bridge. The ability of LytM to target different types of bonds in the peptidoglycan provides it with a broader spectrum of antimicrobial activity compared to LSS.
In summary, the substrate specificities of LSS and LytM influence their antimicrobial potential by determining the types of bonds they can cleave in the peptidoglycan structure of S. aureus. This specificity allows them to target specific components of the bacterial cell wall, making them effective antimicrobial agents against different strains of S. aureus with varying peptidoglycan compositions.

How can the understanding of LSS and LytM substrate specificities be leveraged to develop novel antimicrobial strategies targeting the bacterial cell wall?

The detailed understanding of the substrate specificities of LSS and LytM provides valuable insights that can be leveraged to develop novel antimicrobial strategies targeting the bacterial cell wall, particularly in the context of combating antibiotic-resistant S. aureus infections. Here are some ways in which this knowledge can be utilized:

Targeted Drug Design: The specific substrate preferences of LSS and LytM can guide the design of targeted antimicrobial drugs that exploit these enzymes' activities. By developing compounds that mimic the preferred substrates of LSS and LytM, it is possible to create inhibitors that disrupt the enzymes' function, leading to bacterial cell lysis and death.

Combination Therapies: Understanding the differences in substrate specificities between LSS and LytM can inform the development of combination therapies that target multiple components of the bacterial cell wall. By utilizing inhibitors or activators that target both enzymes simultaneously, synergistic effects can be achieved, enhancing the overall antimicrobial efficacy.

Precision Medicine: Tailoring antimicrobial treatments based on the specific peptidoglycan composition of the infecting S. aureus strain can lead to more personalized and effective therapies. By matching the substrate specificities of LSS and LytM inhibitors to the unique characteristics of the bacterial cell wall, precision medicine approaches can be developed to combat antibiotic-resistant infections.

Drug Resistance Prevention: Exploiting the substrate specificities of LSS and LytM can help in designing strategies to prevent the development of drug resistance. By targeting specific bonds in the peptidoglycan structure that are essential for bacterial survival, it is possible to minimize the likelihood of resistance mechanisms emerging.

In conclusion, the in-depth knowledge of LSS and LytM substrate specificities opens up new avenues for the development of innovative antimicrobial strategies that target the bacterial cell wall. By leveraging this understanding, researchers can design more effective and tailored approaches to combat S. aureus infections and address the challenge of antibiotic resistance.