title: Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase.
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.
authors.affiliation: Department of Chemistry and Biochemistry, Montclair State University, 1 Normal Avenue, Montclair, NJ, 07043, USA.
authors.forename: Romy
authors.initials: R
authors.lastname: Perez-Abraham
authors.affiliation: Department of Chemistry and Biochemistry, Montclair State University, 1 Normal Avenue, Montclair, NJ, 07043, USA.
authors.forename: Karla Garabiles
authors.initials: KG
authors.lastname: Sanchez
authors.affiliation: Department of Chemistry and Biochemistry, Montclair State University, 1 Normal Avenue, Montclair, NJ, 07043, USA.
authors.forename: Melany
authors.initials: M
authors.lastname: Alfonso
authors.affiliation: Department of Chemistry and Biochemistry, Montclair State University, 1 Normal Avenue, Montclair, NJ, 07043, USA.
authors.forename: Ueli
authors.initials: U
authors.lastname: Gubler
authors.affiliation: Department of Chemistry and Biochemistry, Montclair State University, 1 Normal Avenue, Montclair, NJ, 07043, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Siekierka
authors.affiliation: Department of Chemistry and Biochemistry, Montclair State University, 1 Normal Avenue, Montclair, NJ, 07043, USA. Electronic address: goodeyn@mail.montclair.edu.
authors.forename: Nina M
authors.initials: NM
authors.lastname: Goodey
mesh_terms: D000818:Animals; D017178:Brugia malayi / Q000201:enzymology / Q000235:genetics*; D002384:Catalysis; D015870:Gene Expression; D015801:Helminth Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D015801:Helminth Proteins; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase
keywords: Antifolate inhibitors; Brugia malayi; Dihydrofolate reductase; Filariasis; Steady-state kinetics