Mimicking enzyme evolution by generating new (βα)8-barrels from (βα)4-half-barrels
Mimicking enzyme evolution by generating new (βα)8-barrels from (βα)4-half-barrels
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DOI：
10.1073/pnas.0405832101
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发表时间：
2004-11-23
影响因子：
11.1
通讯作者：
Sterner, R
中科院分区：
文献类型：
--
作者：
Hocker, B;Claren, J;Sterner, R
基因的复制和融合是酶进化的重要机制，它们使蛋白质的功能结构域发生增殖和连接。氨基酸序列和三维结构的分析表明，（β α）（8）-桶，这是酶中最常见的折叠，已经通过（β α）（4）-半桶结构域的复制，融合和混合而进化。在这里，我们通过从咪唑甘油磷酸合酶（HisF）和N '[（5'-磷酸核糖基）甲亚胺基]-5-氨基咪唑-4-甲酰胺-核糖核苷酸异构酶（HisA）推导的（β α）（4）-半桶体外产生（β α）（8）-桶来模拟这种进化策略。为此，HisF的C-末端（β α）（4）-半桶（HisF-C）的基因被复制并串联融合以产生HisF-CC，其比HisF-C更稳定。在下一步中，通过优化HisF-CC的β-桶中心内的侧链相互作用，产生单体和紧凑的（β α）（8）-桶蛋白HisF-C*C。此外，将HisF和HisA的N-和C-末端（β α）（4）-半桶的基因双融合以产生嵌合蛋白HisFA和HisAF。尽管HisFA含有天然二级结构元件但采用不明确的缔合状态，但（β α）（8）-桶HisAF是一种稳定且紧凑的单体，其可逆地以高协同性展开。获得的结果表明，以前未描述的酶活性多样化的维度：新的（β α）（8）-桶与新的功能可能已经演变的（β α）（4）-半桶结构域与不同的功能特性的交换。
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.