Establishing catalytic activity on an artificial (βα)8-barrel protein designed from identical half-barrels.

It has been postulated that the ubiquitous (βα)8-barrel enzyme fold has evolved by duplication and fusion of an ancestral (βα)4-half-barrel. We have previously reconstructed this process in the laboratory by fusing two copies of the C-terminal half-barrel HisF-C of imidazole glycerol phosphate synthase (HisF). The resulting construct HisF-CC was stepwise stabilized to Sym1 and Sym2, which are extremely robust but catalytically inert proteins. Here, we report on the generation of a circular permutant of Sym2 and the establishment of a sugar isomerization reaction on its scaffold. Our results demonstrate that duplication and mutagenesis of (βα)4-half-barrels can readily lead to a stable and catalytically active (βα)8-barrel enzyme.