Inhibition of Staphylococcus aureus PriA Helicase by Flavonol Kaempferol - PubMed (original) (raw)
Inhibition of Staphylococcus aureus PriA Helicase by Flavonol Kaempferol
Yen-Hua Huang et al. Protein J. 2015 Jun.
Abstract
Staphylococcus aureus is an important etiological agent responsible for healthcare-associated infections. In this study, the effect of flavonoids on the inhibition of S. aureus PriA (SaPriA), an essential helicase for DNA replication restart, which is critical for bacterial survival, was investigated. Using vanadate-sensitive colorimetric assay, the concentration of phosphate, from ATP hydrolysis by SaPriA, was decreased to 37 and 69%, respectively, in the presence of 35 μM kaempferol and myricetin. The effect of quercetin, galangin, dihydromyricetin, and myricitrin was insignificant. From titration curve, IC50 of kaempferol for SaPriA was determined to be 22 ± 2 μM. Using fluorescence quenching, we identified that kaempferol can bind to SaPriA with K(d) of 9.1 ± 3.2 μM. To our knowledge, these preliminary results constituted the first study regarding that naturally occurring product such as flavonols kaempferol and myricetin can be potent inhibitors targeting PriA.
Figures
Fig. 1
SDS-PAGE of the purified SaPriA and molecular mass standards
Fig. 2
Molecular structure of myricetin, quercetin, kaempferol, and galangin
Fig. 3
Inhibition of the ATPase activity of SaPriA. The ATPase activity of purified SaPriA was analyzed using the vanadate-sensitive colorimetric assay. The ATPase activity assay (2 mL of reaction volume) for SaPriA (2 μM) was analyzed in 20 mM HEPES (pH 7.0), 5 mM of MgCl2, 1 mM of ATP, and 35 μM of flavonoid for 2 h, and then the OD610 was analyzed. The reaction mixture with (lane 1) or without SaPriA (lane 2) was analyzed. The reaction mixture with SaPriA was analyzed in the presence of kaempferol (lane 3), myricetin (lane 4), dihydromyricetin (lane 5), galangin (lane 6), quercetin (lane 7), and myricitrin (lane 8)
Fig. 4
The titration curve of kaempferol for SaPriA. IC50 of kaempferol was determined to be 22 ± 2 μM using graphic analysis
Fig. 5
The fluorescence quenching of SaPriA by kaempferol. Compound concentrations, from the top down, are 0 to 10 μM. Fluorescence titration was performed using a spectrofluorimeter (Hitachi F-2700). An aliquot amount of kaempferol was added into the solution containing SaPriA (0.5 μM), 20 mM HEPES, and 100 mM NaCl at pH 7.0. The K d value was obtained by the equation: ΔF = ΔFmax − K d(ΔF/[compound]) (Enzyme Kinetics module of Sigma-Plot)
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