De Novo Disulfide Bridges Cause Pleiotropic Effects on Phytase Characteristics Other Than Stability

De novo disulfide bridges have been documented to increase the thermostability of proteins, but little attention has been devoted to their effect on catalytic kinetics. We previously attempted to improve the stability of Escherichia coli AppA2 phytase through creation of nine de novo disulfide bridge mutants. However, only Q134C/A202C marginally increased the stability, while seven mutants showed decreased catalytic efficiency. Moreover, at 37°C, ss2‐ (C133/C408S), ss3‐ (C178S/C188) and A21C/W40C maintained the same thermostability as WT while showing decreased activity with altered pH profile and optimal temperature. We explored the mechanism of these effects through additional mutations in one mutant, T33C/L170C. Both T33C/L170C/L217M, with a mutation to counteract atom displacement by the de novo disulfide bridge, and T33C/L170C/A25insG, with a mutation to counteract decreased atom motility, had a higher (P ≤ 0.05) Vmax, kcat and kcat/Km than T33C/L170C at 37°C. These results suggest that de novo disulfide bridges in AppA2 phytase decreased catalytic efficiency partially through alterations in atom motility and displacement of atom coordinates. Research supported by Cornell CAT grant.