Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase

Romy Perez-Abraham, Karla Garabiles Sanchez, Melany Alfonso, Ueli Gubler, John J. Siekierka, Nina M. Goodey

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity kcat was found to be 1.4 ± 0.1 s−1, the Michaelis Menten constant KM for dihydrofolate 14.7 ± 3.6 μM, and the equilibrium dissociation constant KD for NADPH 25 ± 24 nM. For BmDHFR, IC50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 μM for trimethoprim, 109 ± 34 μM for pyrimethamine, 154 ± 46 μM for 2,4-diaminoquinazoline, 771 ± 44 μM for cycloguanil, and >20,000 μM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR.

Original languageEnglish
Pages (from-to)81-85
Number of pages5
JournalProtein Expression and Purification
Volume128
DOIs
StatePublished - 1 Dec 2016

Keywords

  • Antifolate inhibitors
  • Brugia malayi
  • Dihydrofolate reductase
  • Filariasis
  • Steady-state kinetics

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