Active-site design of cofactor-free enzymes

Enzymes are the catalysts of Nature. Their mild reactions conditions and high selectivity make them ideal tools for organic synthesis. Many biocatalysts, however, are characterized by limited substrate spectra. This requires methods for the modification of enzymatic mechanisms in order to create new reactivities. The breaking and formation of bonds between carbon atoms is of high interest in view of synthetic applications. However, these reactions are very challenging for biocatalysis. The bacterial enzymes arylmalonate decarboxylase and eudesmol synthase catalyse these reactions with outstanding selectivity. The molecular mechanisms of this selectivity lie in the selective stabilization of reaction intermediates on the one hand, and in targeted quenching of these intermediates on the other hands. On basis of mechanistic studies and computational simulations, we aim to incorporate functional groups at specific positions in order to direct the reactions of both enzymes towards the formation of new products. With the current knowledge, it is extremely difficult to formulate precise predictions on the outcome of any modification of the active site. Therefore, a targeted randomization of sets of amino acids in the active site coupled with a high-throughput screening of selected variants is considered to be the most promising strategy to influence the product formation of both enzymes. For the precise modification of interactions between substrate and protein we plan to incorporate functional groups that are not present in natural enzymes. For this, we will incorporate unnatural amino acids. The approach of Active Site Design is the targeted introduction of novel functional group coupled to a simultaneous variation of the molecular context. The optimal integration of the unnatural amino acids is aimed to achieve novel reactivities. Key for this proceeding is an interdisciplinary collaboration between enzyme engineering, organic synthesis and mechanistic studies. 1

The project "Active site design" investigated how enzymes can cleave carbon-carbon bonds without the aid of organic cofactors. It focused on the mechanistic understanding and engineering of the bacterial arylmalonate decarboxylase, that can convert simple starting materials into chiral products without using toxic metals. A key question was how this enzyme controls which mirror-image product is formed. Surprisingly, we discovered the newly designed enzyme variant operates via a different mechanistic pinricple, in which bond breakting and bond making occur simultaneously. With the insights gained from these studies we could extend the applicability of this enzyme to other substrates. In addition, we explored the evolutionary origins of this enzyme family. By reconstructing ancestral versions of arylmalonate decarboxylase, we obtained enzyme variants with improved robustness and stereoselectivity. Together, the project delivers both fundamental understanding and practical indications: it explains how engineered enzymes can change binding modes and stereochemical pathways, and it demonstrates that ancestral reconstruction can provide stable, high-performing starting points for future biocatalysts. This contributes to greener routes for producing chiral building blocks that are important for multiple technology sectors.