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

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

doi:10.3389/fchem.2024.1415644

N^α-acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification

Journal Article · Thu Jul 11 04:00:00 EDT 2024 · Frontiers in Chemistry

DOI:https://doi.org/10.3389/fchem.2024.1415644· OSTI ID:2470259

Kelley, Emma H. ^[1]; Osipiuk, Jerzy ^[2]; Korbas, Malgorzata ^[3]; Endres, Michael ^[4]; Bland, Alayna ^[1]; Ehrman, Victoria ^[1]; Joachimiak, Andrzej ^[2]; Olsen, Kenneth W. ^[1]; Becker, Daniel P. ^[1]

Loyola University Chicago, IL (United States)
Argonne National Laboratory (ANL), Argonne, IL (United States); Univ. of Chicago, IL (United States)
Univ. of Saskatchewan, Saskatoon, SK (Canada). Canadian Light Source, Inc.
Univ. of Chicago, IL (United States)

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^α -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 N⁵, N⁵-di-methyl N^α-acetyl-L-ornithine (k_cat/K_m = 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; K_i = 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 K_i = 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).

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS); Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-06CH11357; SC0012704

OSTI ID:: 2470259

Journal Information:: Frontiers in Chemistry, Journal Name: Frontiers in Chemistry Vol. 12; ISSN 2296-2646

Publisher:: Frontiers Research FoundationCopyright Statement

Country of Publication:: United States

Language:: English

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

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