Fig. 1: Effect of methionine substitution of yeast cytosine deaminase.
A Enzymatic activities of the purified his-tagged yCD and its mutant proteins at 37 °C in PBS. 250 nM of the protein was incubated with 1 mM 5-FC substrate, and the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) was monitored by measuring the absorbance at 276 nm and 266 nm, which was used to derive the % of 5-FU produced (n = 3). B Thermal stability of the purified wild-type yCD and its mutant proteins assessed by circular dichroism spectroscopy at different temperatures. The readings were recorded at 220 nm from 25 °C to 85 °C and the measurements were used in determining the percentage of folded protein. The melting temperature (Tm) was determined using GraphPad Prism software. C The oxidation resistance abilities of wild-type yCD and mutant yCD-M100H were assessed by pre-incubating 500 nM enzymes in PBS with hydrogen peroxide (H2O2) at concentrations ranging from 7.84 mM to 4.90 M for 10 min. After the addition of catalase to remove residual H2O2, the residual enzymatic activities were determined using 1 mM 5-FC normalized to the corresponding enzymes without hydrogen peroxide treatment (n = 3). Data were shown as mean ± SD.
