Design and evolution of chimeric streptavidin for protein-enabled dual gold catalysis
Design and evolution of chimeric streptavidin for protein-enabled dual gold catalysis
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DOI：
10.1038/s41929-021-00651-9
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发表时间：
2021-08-02
期刊：
影响因子：
37.8
通讯作者：
Ward, Thomas R.
中科院分区：
文献类型：
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作者：
Christoffel, Fadri;Igareta, Nico, V;Ward, Thomas R.
人工金属酶是通过将有机金属催化剂固定在可进化的蛋白质支架中而产生的。由于链霉亲和素具有二聚体的四元结构，可以精确定位两个金属辅因子以激活单个底物，从而扩大了人造金属酶的反应范围。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的双金活化的人工氢氨酶方面所做的努力。在模型的指导下，我们设计了一个嵌合的链霉亲和素，它带有一个疏水的盖子来屏蔽它的活性部位，这加强了两个协同生物素化的金辅因子的有利定位。利用大肠杆菌无细胞提取物进行了三轮定向进化，发现了有利于抗Markovnikov产物(具有96%区域选择性的吲哚甲酰胺，51个转换数)和Markovnikov产物(具有99%区域选择性的苯基二氢喹唑酮，333个转换数)的突变体。
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 sigma,pi-activation of an ethynylphenylurea substrate, or the Markovnikov product (a phenyl-dihydroquinazolinone with 99% regioselectivity, 333 turnover numbers), resulting from the pi-activation of the alkyne by gold.