Ledesma Fernández, AlbaVelasco Lozano, SusanaCampos Muelas, PedroMadrid González, RicardoLópez Gallego, FernandoCortajarena, Aitziber L.2024-07-022024-07-022024-04-12Ledesma-Fernandez A,Velasco-Lozano S, Campos-Muelas P, Madrid R,L opez-Gallego F, Cortajarena AL. Engineering bio-brick protein scaffolds for organizing enzymeassemblies. Protein Science. 2024;33(5):e4984.0961-83681469-896X10.1002/pro.4984https://hdl.handle.net/20.500.14352/105453Enzyme 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.engEngineering bio‐brick protein scaffolds for organizing enzyme assembliesInglésjournal articlehttps://doi.org/10.1002/pro.4984https://pubmed.ncbi.nlm.nih.gov/38607190/restricted access577.11576Consesus tetratricopeptide repeats (CTPRs)Metal‐driven assemblyMulti‐enzymatic cascadesMulti‐enzyme systemsProtein engineeringProtein scaffoldsProtein self‐assemblyBioquímica (Biología)Biología molecular (Biología)Biotecnología2403 Bioquímica2415 Biología Molecular