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.\xa0 Frequently tradeoffs are observed, where improvements in some features can come at the expense of others.\xa0 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. \xa0Biomimetic polyethylene glycol (PEG) swing arms modified with NAD(H) have been explored to introduce synthetic swing arms into fused oxidoreductases.\xa0 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.\xa0 The modular assembly approach enables the K M values of new enzymes to be predictable, based on the parental enzymes.\xa0 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.