Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

• Main Authors: Charlotte Kürten, Bengt Carlberg, Per-Olof Syrén
• Format: Article
• Language: English
• Published: MDPI AG 2016-06-01
• Series: Catalysts
• Subjects: enzyme promiscuity; enzyme catalysis; biocatalysis; reaction mechanisms; molecular modeling; amidase; esterase
• Online Access: http://www.mdpi.com/2073-4344/6/6/90

The discovery and generation of biocatalysts with extended catalytic versatilities are of immense relevance in both chemistry and biotechnology. An enhanced atomistic understanding of enzyme promiscuity, a mechanism through which living systems acquire novel catalytic functions and specificities by evolution, would thus be of central interest. Using esterase-catalyzed amide bond hydrolysis as a model system, we pursued a simplistic in silico discovery program aiming for the identification of enzymes with an internal backbone hydrogen bond acceptor that could act as a reaction specificity shifter in hydrolytic enzymes. Focusing on stabilization of the rate limiting transition state of nitrogen inversion, our mechanism-guided approach predicted that the acyl hydrolase patatin of the α/β phospholipase fold would display reaction promiscuity. Experimental analysis confirmed previously unknown high amidase over esterase activity displayed by the first described esterase machinery with a protein backbone hydrogen bond acceptor to the reacting NH-group of amides. The present work highlights the importance of a fundamental understanding of enzymatic reactions and its potential for predicting enzyme scaffolds displaying alternative chemistries amenable to further evolution by enzyme engineering.