Título
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
Autor
ELIEL RAFAEL ROMERO GARCIA
ALFREDO TELLEZ VALENCIA
MARIA FATIMA TRUJILLO ESQUIVEL
JOSE GUADALUPE SAMPEDRO PEREZ
HUGO NAJERA PEÑA
ARTURO ROJO DOMINGUEZ
J DE JESUS GARCIA SOTO
Nivel de Acceso
Acceso Abierto
Referencia de publicación
ISSN/1110-7243
Referencia de datos
doi: http://downloads.hindawi.com/journals/bmri/2009/201075.pdf
Resumen o descripción
An aprE mutant from B. subtilis 168 lacking the connecting loop Leu75–Leu82 which is predicted to encode a Ca2+ binding site was
constructed. Expression of the mutant gene (aprEΔLeu75–Leu82) produced B. subtilis colonies lacking protease activity. Intrinsic
fluorescence analysis revealed spectral differences between wild-type AprE and AprEΔL75–L82. An AprEΔL75–L82 variant with
reestablished enzyme activity was selected by directed evolution. The novel mutations Thr66Met/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 connects β-sheet e3 with α-helix c plays a structural
role on enzyme activity of AprE from B. subtilis 168.
Journal of Biomedicine and Biotechnology
Editor
London : Hindawi Publishing Corporation
Fecha de publicación
8 de julio de 2020 8 de julio de 2020 2009
Tipo de publicación
Artículo
Recurso de información
http://ilitia.cua.uam.mx:8080/jspui/handle/123456789/608
Journal of Biomedicine and Biotechnology, vol., 2009, núm. 201075, aug, 2009
Formato
application/pdf
Idioma
Inglés
Repositorio Orígen
Concentración de Recursos de Información Científica y Académica, UAM Cuajimalpa
Descargas
0