%0 Journal Article
%T Engineering bio-brick protein scaffolds for organizing enzyme assemblies.
%A Ledesma-Fernandez A
%A Velasco-Lozano S
%A Campos-Muelas P
%A Madrid R
%A López-Gallego F
%A Cortajarena AL
%J Protein Sci
%V 33
%N 5
%D 2024 May
%M 38607190
%F 6.993
%R 10.1002/pro.4984
%X Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.