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

Abstract

Abstract: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.

Separating Thermodynamics from Kinetics—A New Understanding of the Transketolase Reaction

“…Creating novel interactions between an active‐site residue and a desired substrate should include a network of hydrogen bonds . As was shown, this is an effective strategy to increase the substrate's affinity towards the active site, although a polarity‐based analysis would suggest the opposite.…”

“…[24] Conclusions Creating novel interactions between an active-site residue and ad esired substrate should include an etwork of hydrogen bonds. [25][26][27][28][29] As was shown, this is an effective strategy to increase the substrate's affinity towards the active site, although ap olarity-based analysisw ould suggest the opposite. This alternative approach for the rational mutagenesis of transketolases towards hydrophobic substrates was demonstrated.…”

An Extremely Sensitive Ultra-High Throughput Growth Selection Assay for the Identification of Amidase Activity

“…In cases where a novel activity must be created from an inactive scaffold, the combination of ultra-high throughput and extreme sensitivity would be of great advantage, since initial variants with slightly improved activity could then be identified. This is the case in projects exploring the promiscuous activity of an enzyme, like the identification of amidase activity of promiscuous esterases for pharmaceutical or chemical industrial applications (Kourist et al 2008 ; Jung et al 2013 ; Hackenschmidt et al 2014 ; Kürten et al 2016 ; Galmés et al 2020 ). A similar application is the engineering of polyester hydrolases (polyesterases) to degrade related polymers like polyamides (Biundo et al 2019 ).…”