Overexpression, Purification, and Biochemical Characterization of the vanC2 d-Ala-d-Ser Ligase from Enterococcus casseliflavus SSK and Its Inhibition by an Oxadiazole Derivative

TL;DR

This study explores a bacterial enzyme linked to vancomycin resistance and shows that a specific chemical compound can inhibit its activity, offering a potential new treatment for resistant infections.

Contribution

The study reports the biochemical characterization of vanC2 EcfDdls and demonstrates inhibition by a novel oxadiazole derivative.

Findings

The oxadiazole derivative CID 45805715 completely inhibited the purified EcfDdls enzyme.

CID 45805715 had an IC50 of 76.7 μM, significantly lower than the reference compound DCS at 313 μM.

The compound also showed antimicrobial activity against vancomycin-resistant E. casseliflavus strain SSK.

Abstract

The bacterial cell wall and enzymes involved in peptidoglycan
biosynthesis
are prime targets for the discovery of novel antibacterial agents.
Among these enzymes, d-alanine-d-alanine ligases
(Ddl) are particularly significant due to their utilization of specific
substrates (d-amino acids) essential for bacterial viability.
Isozymes of Ddl that utilize alternative substrates such as d-lactate or d-serine are found in vancomycin-resistant Gram-positive
bacteria, initially identified in Enterococcus species, and now represent
a growing concern in clinical settings. In this study, we isolated
and identified vancomycin-resistant Enterococcus casseliflavus (E. casseliflavus) strain SSK and
used it for amplification, cloning, and purification of the vanC2
type of d-alanine-d-serine ligase (EcfDdls). Investigations
of substrate specificity and enzyme kinetics provided insights into
the enzyme’s mechanistic action. Evaluation of the inhibitory
potential of the previously virtually screened oxadiazole derivative
1-[(5-methyl-1,2-oxazol-3-yl)methyl]-4-{[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]methyl}piperazine
(CID 45805715) was carried out using an inorganic phosphate detection
assay, which demonstrated complete enzymatic inhibition of purified
EcfDdls. When tested, CID 45805715 significantly inhibited activity
of Ddl, with an IC50 of 76.7 μM, compared to 313
μM for the reference compound DCS. Moreover, this compound also
exhibited antimicrobial activity against vancomycin-resistant E. casseliflavus strain SSK. Thus, these findings
provide valuable insights into the activity and inhibition of vanC2
EcfDdls, offering a promising avenue for addressing vancomycin resistance
in enterococci, particularly in nosocomial infections affecting immunocompromised
patients.

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