TY - JOUR
T1 - Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase
AU - Perez-Abraham, Romy
AU - Sanchez, Karla Garabiles
AU - Alfonso, Melany
AU - Gubler, Ueli
AU - Siekierka, John J.
AU - Goodey, Nina M.
N1 - Publisher Copyright:
© 2016 Elsevier Inc.
PY - 2016/12/1
Y1 - 2016/12/1
N2 - 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.
AB - 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.
KW - Antifolate inhibitors
KW - Brugia malayi
KW - Dihydrofolate reductase
KW - Filariasis
KW - Steady-state kinetics
UR - http://www.scopus.com/inward/record.url?scp=84983385721&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2016.08.012
DO - 10.1016/j.pep.2016.08.012
M3 - Article
C2 - 27544923
AN - SCOPUS:84983385721
SN - 1046-5928
VL - 128
SP - 81
EP - 85
JO - Protein Expression and Purification
JF - Protein Expression and Purification
ER -