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

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