Inhibition of Stenotrophomonas maltophilia dihydrofolate reductase by methotrexate: A single slow-binding process

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 with an association rate constant (k_i) of 3.8 × 10⁷ M^{− 1}s^{− 1}. Possible mechanisms for MTX binding to S. maltophilia DHFR are discussed.

Keywords::

• Stenotrophomonas maltophilia
• dihydrofolate reductase
• methotrexate
• antifolates
• slow-binding inhibitors

Acknowledgements

This work was supported in part by a grant from PBL International to J.N.R-L and from Fondo de Investigaciones Sanitarias (FIS) (Project 01/3025) and Red Temática de Investigación Cooperativa de Centros del Cáncer (RTICCC) (Red de centros C03/10) to J.C-H. M.D.N-M has a fellowship from Fundación Séneca, Murcia, Spain. We thank Dr. Joaquín Ruiz-Gómez at the Servicio de Microbiología del Hospital Clínico Universitario Virgen de la Arrixaca for providing us with the S. maltophilia clinical isolates.