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Updated: Jul 11, 2026

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
Inhibition of Stenotrophomonas maltophilia dihydrofolate reductase by methotrexate: a single slow-binding process
M D Navarro-Martínez1, J Cabezas-Herrera, F García-Cánovas
1Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular A, Facultad de Biología, Universidad de Murcia, E-301 00 Espinardo, Murcia, Spain.
Abstract:
Although antifolates such as trimethoprim are used in the clinical treatment of Stenotrophomonas maltophilia infection, the dihydrofolate reductase (DHFR) of this microorganism is scarcely known because it has never been isolated. Here, we describe the purification of this enzyme and kinetically characterize its inhibition by methotrexate (MTX). Upon MTX treatment, time-dependent, slow-binding inhibition was observed due to the generation of a long-lived, slowly dissociating enzyme-NADPH-inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (K(I) = 28.9 +/- 1.9 pM) with an association rate constant (k(i)) of 3.8 x 10(7) M(-1)s(-1). Possible mechanisms for MTX binding to S. maltophilia DHFR are discussed.
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