Engineering and Directed Evolution of aCa2+Binding Site A-Deficient AprE Mutant Reveal an Essential Contribution of the LoopLeu75–Leu82to Enzyme Activity

摘要

AnaprEmutant fromB. subtilis168 lacking the connecting loopLeu75–Leu82which is predicted to encode aCa2+binding site was constructed. Expression of the mutant gene (aprEΔLeu75–Leu82) producedB. subtiliscolonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprEΔL75–L82. An AprEΔL75–L82variant with reestablished enzyme activity was selected by directed evolution. The novel mutationsThr66Met/Gly102Asp located in positions which are predicted to be important for catalytic activity were identified in this variant. Although these mutations restored hydrolysis, they had no effect with respect to thermal inactivation of AprEΔL75–L82 T66M G102D. These results support the proposal that in addition to function as a calcium binding site, the loop that connectsβ-sheet e3 withα-helix c plays a structural role on enzyme activity of AprE fromB. subtilis168.