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Expression, Purification and Enzymatic Characterization of Brugia malayi Dihydrofolate Reductase
Author(s) -
PerezAbraham Romy
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.834.4
Subject(s) - brugia malayi , dihydrofolate reductase , enzyme , biology , affinity chromatography , microbiology and biotechnology , biochemistry , chemistry , filariasis , helminths , zoology
Dihydrofolate reductase (DHFR) is an essential enzyme that plays an important role in the production of cofactors that are required in deoxyribonucleic acid (DNA) synthesis. The literature suggests that DHFR is a potential drug target for the elimination of the parasitic worm, Brugia malayi . Brugia malayi is one of the causative parasites of lymphatic filariasis, a globally neglected tropical disease. In this study, we expressed and purified B. malayi DHFR. Expression was carried out in E. coli with a His 6 ‐tagged construct, and purification was achieved through affinity chromatography using LOBSTR E. coli cells. The resulting purified and active enzyme was used for steady‐state kinetics characterization and Michaelis‐Menten inhibition studies. The catalytic activity, k cat , was found to be 1.4 ± 0.1s −1 , the Michaelis Menten constant, K M , 14.7 ± 3.6μM for dihydrofolate, and the equilibrium dissociation constant, K D , 22 ±0.01μM. B. malayi DHFR was compared to other DHFR homologs in terms of ligand specificity determining residues; L. major, T. cruzi, T. bruci, and T. gondii exhibited the highest homology. Known DHFR inhibitors of these DHFR homologs were assayed with B. malayi DHFR; IC 50 values were determined to be 0.003 ± 0.36 μM for methotrexate, 90.2 ± 7.9 μM for pyrimethamine, 31.8 ± 5.4 μM for trimethoprim, 770.6 ± 534 μM for cycloguanil, >20,000 μM for 2,4‐diaminopyrimidine, and 192.9 ± 88.0 μM for 2,4‐diaminoquinazoline. These results provide the basis for the development of more potent compounds that can inhibit B. malayi DHFR without affecting human DHFR, and help cure lymphatic filariasis.