(719c) Engineering of Aldehyde Dehydrogenases Towards Noncanonical Redox Cofactor Nicotinamide Mononucleotide

Non-canonical redox cofactors play a crucial role in biomanufacturing by enhancing the selectivity, productivity, and cost of biocatalytic processes. However, they have achieved widespread adoption due to challenges in engineering enzymes to accept these cofactors. These challenges primarily derive from the lack of central design principles to guide engineering efforts, that can be applied not only to individual enzymes but across protein families.

To address these challenges, we deployed sequence similarity networks and a high throughput colorimetric assay to screen through representatives of the entire aldehyde dehydrogenase (ALDH) protein family and identify ALDH scaffolds with activity towards the noncanonical redox cofactor nicotinamide mononucleotide (NMN⁺). Over a third of the ALDH’s tested showed detectable activity towards NMN⁺, and stunningly, several of these ALDH’s exhibit kinetic parameters superior to those of engineered NMN⁺ utilizing enzymes. Sequence and structural analyses reveal a conserved motif among these NMN⁺ utilizing ALDH’s that is responsible for improving NMN⁺ binding at the active site by establishing a hydrogen binding network with the cofactor. To further investigate the role of this motif it was incorporated into six diverse ALDH’s, resulting in up to a 60-fold increase in catalytic efficiency towards NMN⁺. Modelling of these enzyme variants suggests that we have identified a structural motif for improving NMN⁺ activity with wide application across the diverse ALDH protein family.

This work demonstrates a high throughput approach for identifying enzymes scaffolds with activity towards non-canonical redox cofactors and highlights the potential for elucidating central design principles to enhance noncanonical redox cofactor activities. These central design principles accelerate engineering efforts and contributes to the growing repertoire of non-canonical cofactor dependent biocatalysts.