(462c) NAD(H)-Peg Swing Arms Improve Both the Activities and Stabilities of Modularly-Assembled Transhydrogenases Designed with Predictable Selectivities

Protein engineering is often used to attempt to enhance the activities, selectivity, and stabilities of enzymes. Tradeoffs are frequently encountered, where improvements in some enzyme features can come at the expense of others. Alternatively, enzymatic activities can be generated through the modular coordination of existing active sites. Substrate channeling mechanisms can be employed to overcome the transport limitations introduced by the use of separate active sites. We have previously shown that synthetic PEG-NAD(H) swing arms can be used to introduce a substrate channeling mechanism between two conjugated, unrelated dehydrogenase enzymes (1). A significant advantage of this approach was that the dehydrogenase active sites remained unmodified, so that the selectivities of the parent active sites were conserved and predictable in the final transhydrogenase. However, the activity of the fused enzyme was low due to a combination of limited electron transfer rates by the swing arms and a limited stability. We have increased the addition of PEG-based NAD(H) swing arms and combined the functions of additional dehydrogenases to create a series of novel, self-contained synthetic transhydrogenases that have no native homologs. We show that the stabilities and the catalytic properties of the resulting transhydrogenases are improved without detriment to the selectivities of the native active sites, emphasizing the functional modularity and predictive nature of this synthetic approach to biocatalyst design.

(1) Ozbakir, H.F., Garcia, K.E., and Banta, S. (2018). Creation of a formate: malate oxidoreductase by fusion of dehydrogenase enzymes with PEGylated cofactor swing arms. Protein Eng Des Sel 31, 103–108