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=12

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=<p>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 <italic>N</italic><sup>ɑ</sup> -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 <italic>N</italic><sup>5</sup>, <italic>N</italic><sup>5</sup>-di-methyl <italic>N</italic><sup>α</sup>-acetyl-L-ornithine (k<sub>cat</sub>/K<sub>m</sub> = 7.32 ± 0.94 × 10<sup>4</sup> M<sup>−1</sup>s<sup>−1</sup>). 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 (IC<sub>50</sub> = 58.7 μM; K<sub>i</sub> = 37.1 ± 0.85 μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC<sub>50</sub> = 50.1 μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against <italic>Escherichia coli</italic> ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril K<sub>i</sub> = 35.9 ± 5.1 μM). The active site structure of di-Zn <italic>Ec</italic>ArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of <italic>E. coli</italic> 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).</p>