AUTHOR=Kelley Emma H. , Osipiuk Jerzy , Korbas Malgorzata , Endres Michael , Bland Alayna , Ehrman Victoria , Joachimiak Andrzej , Olsen Kenneth W. , Becker Daniel P. 

TITLE=Nα-acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification

JOURNAL=Frontiers in Chemistry

VOLUME=Volume 12 - 2024

YEAR=2024

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2024.1415644

DOI=10.3389/fchem.2024.1415644

ISSN=2296-2646

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 ɑacetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE provides a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated, including the synthesis of substrate analog N 5 ,N 5 -di-methyl N  -acetyl-L-ornithine substrate (kcat/Km = 7.32 ± 0.94 x 10 4 M -1 s -1 ). This new assay enables screening of potential inhibitors that absorb in the UV region, and thus is superior to the historic 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-diethylaminophenylboronic 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 report two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two crystal structures published of ArgE (mono-Zn and di-Zn).