Mimicking enzyme evolution by generating new ( )8-barrels from ( )4-half-barrels

Gene duplication and fusion events that multiply and link functional protein domains are crucial mechanisms of enzyme evolution. The analysis of amino acid sequences and three-dimensional structures suggested that the ( )8-barrel, which is the most frequent fold among enzymes, has evolved by the duplication, fusion, and mixing of ( )4-half-barrel domains. Here, we mimicked this evolutionary strategy by generating in vitro ( )8-barrels from ( )4-half-barrels that were deduced from the enzymes imidazole glycerol phosphate synthase (HisF) and N [(5 -phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide isomerase (HisA). To this end, the gene for the C-terminal ( )4half-barrel (HisF-C) of HisF was duplicated and fused in tandem to yield HisF-CC, which is more stable than HisF-C. In the next step, by optimizing side-chain interactions within the center of the -barrel of HisF-CC, the monomeric and compact ( )8-barrel protein HisFC*C was generated. Moreover, the genes for the Nand C-terminal ( )4-half-barrels of HisF and HisA were fused crosswise to yield the chimeric proteins HisFA and HisAF. Whereas HisFA contains native secondary structure elements but adopts ill-defined association states, the ( )8-barrel HisAF is a stable and compact monomer that reversibly unfolds with high cooperativity. The results obtained suggest a previously undescribed dimension for the diversification of enzymatic activities: new ( )8-barrels with novel functions might have evolved by the exchange of ( )4-halfbarrel domains with distinct functional properties.