Engineering and Directed Evolution of a Ca2+ Binding Site A-Deficient AprE Mutant Reveal an Essential Contribution of the Loop Leu75–Leu82 to Enzyme Activity. (20th August 2009)

• Record Type: Journal Article
• Authors: Romero-García, Eliel R.; Téllez-Valencia, Alfredo; Trujillo, María F.; Sampedro, José G.; Nájera, Hugo; Rojo-Domínguez, Arturo; García-Soto, Jesús; Pedraza-Reyes, Mario
• Other Names: Makhatadze George Academic Editor.
• Is Part Of: Journal of biomedicine and biotechnology. Volume 2009(2009)
• Publication Date: 2009-08-20
• Subjects: Medicine -- Periodicals; Biology -- Periodicals; Biotechnology -- Periodicals; Medicine; Biology; Biotechnology; Médecine; Biologie; Biotechnologie; Biology; Biotechnology; Medicine; Biotechnology; Biomedicine; Electronic journals; Periodical; Periodicals; Electronic journals; 610
• DOI: 10.1155/2009/201075
• Languages: English
• ISSNs: 1110-7243

Abstract

Abstract : An aprE mutant from B. subtilis 168 lacking the connecting loopLeu 75 – Leu 82 which is predicted to encode aCa 2 + binding site was constructed. Expression of the mutant gene ( aprE Δ L e u 75 – L e u 82 ) produced B. subtilis colonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprEΔ L 75 – L 82 . An AprEΔ L 75 – L 82 variant with reestablished enzyme activity was selected by directed evolution. The novel mutationsThr 66 Met/Gly 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 75 – L 82 T 66 M G 102 D . 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 from B. subtilis 168.