期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 101, 期 47, 页码 16448-16453出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0405832101
关键词
chimeric proteins; gene duplication; histidine biosynthesis; TIM-barrel
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 (betaalpha)(8)-barrel, which is the most frequent fold among enzymes, has evolved by the duplication, fusion, and mixing of (betaalpha)(4)-half-barrel domains. Here, we mimicked this evolutionary strategy by generating in vitro (betaalpha)(8)-barrels from (betaalpha)(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 (betaalpha)(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 beta-barrel of HisF-CC, the monomeric and compact (betaalpha)(8)-barrel protein HisF-C*C was generated. Moreover, the genes for the N- and C-terminal (betaalpha)(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 (betaalpha)(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 (betaalpha)(8)-barrels with novel functions might have evolved by the exchange of (betaalpha)(4)-half-barrel domains with distinct functional properties.
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