Establishing wild-type levels of catalytic activity on natural and artificial (βα) 8 -barrel protein scaffolds

Publisher Website
Google Scholar
Abstract
The generation of high levels of new catalytic activities on natural and artificial protein scaffolds is a major goal of enzyme engineering. Here, we used random mutagenesis and selection in vivo to establish a sugar isomerisation reaction on both a natural (βα) 8 -barrel enzyme and a catalytically inert chimeric (βα) 8 -barrel scaffold, which was generated by the recombination of 2 (βα) 4 -half barrels. The best evolved variants show turnover numbers and substrate affinities that are similar to those of wild-type enzymes catalyzing the same reaction. The determination of the crystal structure of the most proficient variant allowed us to model the substrate sugar in the novel active site and to elucidate the mechanistic basis of the newly established activity. The results demonstrate that natural and inert artificial protein scaffolds can be converted into highly proficient enzymes in the laboratory, and provide insights into the mechanisms of enzyme evolution.