N α -acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification.
Front Chem
; 12: 1415644, 2024.
Article
en En
| MEDLINE
| ID: mdl-39055043
ABSTRACT
Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express N É -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N 5, N 5-di-methyl N α-acetyl-L-ornithine (kcat/Km = 7.32 ± 0.94 × 104 M-1s-1). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC50 = 58.7 µM; Ki = 37.1 ± 0.85 µM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC50 = 50.1 µM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril Ki = 35.9 ± 5.1 µM). The active site structure of di-Zn EcArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Idioma:
En
Revista:
Front Chem
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Suiza