NAD(H)‐PEG Swing Arms Improve Both the Activities and Stabilities of Modularly‐Assembled Transhydrogenases Designed with Predictable Selectivities

Abstract

Protein engineering has been used to enhance the activities, selectivities, and stabilities of enzymes. Frequently tradeoffs are observed, where improvements in some features can come at the expense of others. Nature uses modular assembly of active sites for complex, multi‐step reactions, and natural “swing arm” mechanisms have evolved to transfer intermediates between active sites. Biomimetic polyethylene glycol (PEG) swing arms modified with NAD(H) have been explored to introduce synthetic swing arms into fused oxidoreductases. Here we report that increasing NAD(H)‐PEG swing arms can improve the activity of synthetic formate:malate oxidoreductases as well as the thermal and operational stabilities of the biocatalysts. The modular assembly approach enables the KM values of new enzymes to be predictable, based on the parental enzymes. We describe four unique synthetic transhydrogenases that have no native homologs, and this platform could be easily extended for the predictive design of additional synthetic cofactor‐independent transhydrogenases.

From Protein Film Electrochemistry to Nanoconfined Enzyme Cascades and the Electrochemical Leaf

“…The activity of the cascade was increased by 2 orders of magnitude compared to the case of freely diffusing cofactor. This concept has been extended to a 2D DNA scaffold and longer linkers …”

Tuning Multistep Biocatalysis through Enzyme and Cofactor Colocalization in Charged Porous Protein Macromolecular Frameworks

“…Because of the high cost of the cofactors for acellular catalysis, recent work has focused on multienzyme systems that replenish the cofactor stock, where a cofactor-regenerating enzyme is coupled with a cofactor-consuming enzyme capable of catalyzing a reaction of interest. , However, these systems still require investment of a large quantity of cofactor to maintain a high overall rate . Tethering cofactors (especially NAD) to different supports (such as nanoparticles or the enzymes themselves) has been a popular approach in the biocatalysis field, as it allows recycling and reuse of cofactor molecules more efficiently. − To further enhance the utilization–regeneration process of cofactor at low quantity, substrate channeling has been proposed in the design of biocatalytic materials. , Compared to conventional multienzyme cascades (no channeling), inducing channeling of a cofactor requires the directed transfer of the cofactor between an enzyme couple without its diffusion into the bulk solution, which can potentially increase the effective local concentration of the cofactor available to the enzymes. As a result, even with a very small input quantity of cofactor, the system can still operate at high catalytic rates.…”