RESUMEN
Natural evolution has produced efficient enzymes of enormous structural diversity. We imitated this natural process in the laboratory to augment the efficiency of an engineered chorismate mutase with low activity and an unusual hexameric topology. By applying two rounds of DNA shuffling and genetic selection, we obtained a 400-fold more efficient enzyme, containing three non-active-site mutations. Detailed biophysical characterization of the evolved variant suggests that it exists predominantly as a trimer in solution, but is otherwise similarly stable as the parent hexamer. The dramatic structural and functional effects achieved by a small number of seemingly innocuous substitutions highlights the utility of directed evolution for modifying protein-protein interactions to produce novel quaternary states with optimized activities.
Asunto(s)
Corismato Mutasa/química , Corismato Mutasa/genética , Corismato Mutasa/metabolismo , Dicroismo Circular , Barajamiento de ADN , Evolución Molecular Dirigida , Biblioteca de Genes , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Desnaturalización Proteica , Estructura Cuaternaria de Proteína , Espectrometría de FluorescenciaRESUMEN
Nature employs a set of 20 amino acids to produce a repertoire of protein structures endowed with sophisticated functions. Here, we combined design and selection to create an enzyme composed entirely from a set of only 9 amino acids that can rescue auxotrophic cells lacking chorismate mutase. The simplified protein captures key structural features of its natural counterpart but appears to be somewhat less stable and more flexible. The potential of a dramatically reduced amino acid alphabet to produce an active catalyst supports the notion that primordial enzymes may have possessed low amino acid diversity and suggests that combinatorial engineering strategies, such as the one used here, may be generally applied to create enzymes with novel structures and functions.