Mimicking Enzyme Evolution by Generating New (β α)8-Barrels from (β α)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 (β α)8-barrel, which is the most frequent fold among enzymes, has evolved by the d...
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Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2004-11, Vol.101 (47), p.16448-16453 |
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Sprache: | eng |
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Zusammenfassung: | 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 (β α)8-barrel, which is the most frequent fold among enzymes, has evolved by the duplication, fusion, and mixing of (β α)4-half-barrel domains. Here, we mimicked this evolutionary strategy by generating in vitro (β α)8-barrels from (β α)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 (β α)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 β-barrel of HisF-CC, the monomeric and compact (β α)8-barrel protein HisF-C*C was generated. Moreover, the genes for the N- and C-terminal (β α)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 (β α)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 (β α)8-barrels with novel functions might have evolved by the exchange of (β α)4-half-barrel domains with distinct functional properties. |
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ISSN: | 0027-8424 1091-6490 |
DOI: | 10.1073/pnas.0405832101 |