Stabilisation and activation of an artificial (beta alpha) (8)-barrel designed from identical (beta alpha) (4)-half-barrels

The striking two-fold sequence and structural symmetry of imidazole glycerol phosphate synthase (HisF) suggests that its (βα)₈-barrel fold has evolved from an ancestral (βα)₄-half-barrel by gene duplication and fusion. We have reconstructed this process in the laboratory by fusing two identical copies of the C-terminal half-barrel HisF-C. The resulting construct HisF-CC was then stabilized by protein engineering to HisF-C*C, HisF-C**C, and HisF-C***C. The analysis of the crystal structure of the artificial (βα)₈-barrel HisF-C***C revealed a striking similarity to wild-type HisF. However, parts at the ends of the fused half-barrels were not visible, impeding rational design of catalytic activity. Subsequent exchange of the non-visible parts by elements from the N-terminal half-barrel HisF-N led to the thermostable and entirely symmetric HisF-sym protein in which all parts of the structure were well defined. HisF-sym and a circularly permuted version were used as scaffolds on which a ligand binding site and a functional active center were established by site-directed mutagenesis and random mutagenesis in combination with selection in vivo, respectively. Our results show that duplication and fusion of a (βα)₄ half-barrel in combination with subsequent mutagenesis readily leads to a stable and catalytically active (βα)₈-barrel, which further supports the proposed evolutionary scenario.