Cooperativity in Escherichia coli L-threonine dehydrogenase and its inhibition by an antibacterial N-pyridylpyrazolone derivative

Obaha, Ana (author), Mikulič Vernik, Nika (author), Mlinar, Karmen (author), Tušek, Marcel (author), Stojkovska, Milena (author), Petek, Nejc (author), Svete, Jurij (author), Novinec, Marko (author)

Antibiotic resistance is an increasing concern in modern healthcare. Therefore, it is important to identify novel antimicrobial agents and new molecular targets for such compounds. Here, we describe the identification of an N-pyridylpyrazolone derivative, 4-(2-aminoethyl)-2-(pyridin-2-yl)-1,2-dihydro-3H-pyrazol-3-one dihydrochloride (compound 1), which is effective against Gram-positive and Gram-negative bacteria and inhibits the enzymatic activity of Escherichia coli L-threonine dehydrogenase (TDH). To characterize its interaction with compound 1, TDH was overexpressed in E. coli. The recombinant enzyme was shown to exist in dilute solution in equilibrium between dimeric and tetrameric forms, with a K$_d$ value for the dimer/tetramer transition of 3 ± 1 nM, and to bind L-threonine cooperatively with a Hill coefficient of 1.4. Compound 1 acted as a partial mixed inhibitor of TDH with an EC$_{50}$ value of 47 ± 16 µM and did not affect the equilibrium between oligomeric states. Altogether, these findings identify compound 1 as a promising starting point for the development of novel antibiotics and as a tool compound for studying the functional properties of TDH.

• Language: English
• Keywords: allostery, enzyme kinetics, partial inhibition, mixed inhibition, homotetramer, antibiotics, mass photometry
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Publication status: Published
• Publication version: Version of Record
• Year: 2025
• Number of pages: 10 str.
• Numbering: Vol. 26, iss. 23, art. 11751
• PID: 20.500.12556/RUL-176702
• UDC: 577.15:547.8
• ISSN on article: 1422-0067
• DOI: 10.3390/ijms262311751
• COBISS.SI-ID: 260536579
• Publication date in RUL: 09.12.2025

Record is a part of a journal

• Title: International journal of molecular sciences
• Shortened title: Int. j. mol. sci.
• Publisher: MDPI
• ISSN: 1422-0067
• COBISS.SI-ID: 2779162

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: alosterija, encimska kinetika, delna inhibicija, mešana inhibicija, homotetramer, antibiotiki, masna fotometrija