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 (␤␣) 4-half-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 HisF-C*C was generated. Moreover, the genes for the N-and 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. chimeric proteins ͉ gene duplication ͉ histidine biosynthesis ͉ TIM-barrel

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 (␤␣) 8-barrel enzyme and a catalytically inert chimeric (␤␣) 8-barrel scaffold, which was generated by the recombination of 2 (␤␣) 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.chimeric protein ͉ enzyme design ͉ enzyme evolution ͉ half barrel ͉ TIM barrel

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