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Design and evolution of chimeric streptavidin for protein-enabled dual gold catalysis
Nature Catalysis ( IF 37.8 ) Pub Date : 2021-08-02 , DOI: 10.1038/s41929-021-00651-9
Fadri Christoffel 1, 2 , Nico V. Igareta 1, 2 , Michela M. Pellizzoni 1 , Boris Lozhkin 1 , Daniel C. Spiess 1, 2 , Ryan L. Peterson 1, 2 , Thomas R. Ward 1, 2 , Laura Tiessler-Sala 3 , Agustí Lledós 3 , Jean-Didier Maréchal 3
Affiliation  

Artificial metalloenzymes result from anchoring an organometallic catalyst within an evolvable protein scaffold. Thanks to its dimer of dimers quaternary structure, streptavidin allows the precise positioning of two metal cofactors to activate a single substrate, thus expanding the reaction scope accessible to artificial metalloenzymes. To validate this concept, we report herein on our efforts to engineer and evolve an artificial hydroaminase based on dual gold activation of alkynes. Guided by modelling, we designed a chimeric streptavidin equipped with a hydrophobic lid shielding its active site, which enforces the advantageous positioning of two synergistic biotinylated gold cofactors. Three rounds of directed evolution using Escherichia coli cell-free extracts led to the identification of mutants favouring either the anti-Markovnikov product (an indole carboxamide with 96% regioselectivity, 51 turnover numbers), resulting from a dual gold σ,π-activation of an ethynylphenylurea substrate, or the Markovnikov product (a phenyl-dihydroquinazolinone with 99% regioselectivity, 333 turnover numbers), resulting from the π-activation of the alkyne by gold.



中文翻译:

用于蛋白质双金催化的嵌合链霉亲和素的设计与进化

人造金属酶是通过将有机金属催化剂锚定在可进化的蛋白质支架内而产生的。由于其二聚体四元结构的二聚体,链霉亲和素允许精确定位两个金属辅因子以激活单个底物,从而扩大了人工金属酶的反应范围。为了验证这一概念,我们在此报告了我们在设计和进化基于炔烃双金活化的人工氢化氨酶的努力。在建模的指导下,我们设计了一种嵌合链霉亲和素,该嵌合链霉亲和素配有一个疏水盖,可保护其活性位点,从而加强了两种协同生物素化金辅助因子的有利定位。使用大肠杆菌的三轮定向进化无细胞提取物导致鉴定出有利于马尔科夫尼科夫产物(一种具有 96% 区域选择性的吲哚甲酰胺,51 个周转数)的突变体,这是由乙炔基苯脲底物的双重金σ、π活化产生的,或马尔科夫尼科夫产物(一种苯基-二氢喹唑啉酮,具有 99% 的区域选择性,333 个周转数),由金对炔烃的π活化产生。

更新日期:2021-08-02
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