Table 1.
Primers used for site-directed mutagenesis.
Figure 1.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Coomassie blue stained 12% SDS-PAGE gel of the purified protein of hAS3MT mutants.
Figure 2.
Catalytic capacity of the hAS3MT mutants.
Reaction mixtures (100 µl) containing 11 µg enzymes, 1 µM iAs3+, 1 mM SAM and 7 mM GSH in PBS (25 mM, pH 7.0) were incubated at 37°C for 2 h with H2O2 treatment before analyzed by HPLC-ICP-MS. The percents of arsenic species (iAs/TAs, MMA/TAs and DMA/TAs) and the two indices (FMR and SMR) of mutants Y58A, G78A, S79A, M103A, Q107A, I136A and E137A are shown in Figure 2a and 2b. Values are the averages ±S.D. of three independent experiments performed by three independently purified proteins.
Figure 3.
A: Substrate concentration dependence of rate.
The lines show the least squares fit of Eq. (1) to the data. B: Double reciprocal plots of the arsenic methylation rate against the concentration of iAs3+. Reaction mixtures (100 µl) containing 11 µg enzymes, 1 mM SAM and 7 mM GSH in PBS (25 mM, pH 7.0) were incubated with different concentrations of iAs3+ at 37°C for 2 h with H2O2 treatment before analysis. Values are the averages ± S.D. of three independent experiments performed by three independently purified proteins.
Table 2.
Kinetic parameters of arsenic methylation for the five mutants S79A, M103A, Q107A, I136A and E137A.
Figure 4.
Double reciprocal plots of the arsenic methylation rate versus the concentration of SAM.
Reaction mixtures (100 µl) containing 11 µg enzymes, 1 µM iAs3+, 7 mM GSH in PBS (25 mM, pH 7.0) were incubated with different concentrations of SAM for 2 h with H2O2 treatment before analysis. Values are the averages ± S.D. of three independent experiments performed by three independently purified proteins.
Figure 5.
CD spectra of hAS3MT and the mutants.
Spectra were taken at the protein concentration of 2 µM at room temperature. Plot is the representative of three independent measurements performed by three independently purified proteins.
Table 3.
Secondary structures of WT-hAS3MT and the mutants estimated from CD spectra.
Figure 6.
Curve-fitted amide I region of the mutants.
The component peaks are the result of curve-fitting using a Gaussian shape. The solid lines represent the experimental FTIR spectra after Savitzky-Golay smoothing, and the dashed lines represent the fitted components. Plot is the representative of three independent measurements carried out by three independently purified proteins.
Table 4.
Secondary structures of WT-hAS3MT and the mutants estimated from ATR-FTIR spectroscopy.
Figure 7.
Interaction modes between SAM, WT-hAS3MT and mutants (Y59A, G78A, S79A, M103A, Q107A, I136A and E137A).
Only the residues 5.0 Å around SAM are displayed.
Table 5.
Residues 5.0 Å around SAM based on the models of WT and mutants Y59A, G78A, S79A, M103A, Q107A, I136A and E137A.
Figure 8.
The model of WT-hAS3MT with SAM.
Only residues in hAS3MT forming potential hydrogen bond network around SAM are presented.