Engineering and directed evolution of a [Ca.sup.2+] binding site A-deficient AprE mutant reveal an essential contribution of the loop [Leu.sub.75]-[Leu.sub.82] to enzyme activity

An aprE mutant from B. subtilis 168 lacking the connecting loop [Leu.sub.75]-[Leu.sub.82] which is predicted to encode a [Ca.sup.2+] binding site was constructed. Expression of the mutant gene (aprEΔ[Leu.sub.75]-[Leu.sub.82])produced B. subtilis colonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprEΔ[L.sub.75]-[L.sub.82]. An AprEΔ[L.sub.75]-[L.sub.82] variant with reestablished enzyme activity was selected by directed evolution. The novel mutations [Thr.sub.66]Met/[Gly.sub.102]Asp 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 AprEΔ[L.sub.75]-[L.sub.82] [T.sub.66]M [G.sub.102]D. These results support the proposal that in addition to function as a calcium binding site, the loop that connects β-sheet e3 with α- helix c plays a structural role on enzyme activity of AprE from B. subtilis 168.