Structure and evolutionary trace-assisted screening of a residue swapping the substrate ambiguity and chiral specificity in an esterase.
Structure and evolutionary trace-assisted screening of a residue swapping the substrate ambiguity and chiral specificity in an esterase.
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DOI：
10.1016/j.csbj.2021.04.041
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发表时间：
2021
影响因子：
6
通讯作者：
Sanz-Aparicio J
中科院分区：
文献类型：
--
作者：
Cea-Rama I;Coscolín C;Katsonis P;Bargiela R;Golyshin PN;Lichtarge O;Ferrer M;Sanz-Aparicio J
一些酶进化出了控制特异性的合适位置。这些位置可以追溯到进化。工程底物混杂但立体特异性水解酶是可行的。我们对具有高底物模糊性的酶的理解仍然有限，因为它们的大活性位点允许底物对接自由到似乎与立体特异性不相容的程度。一种可能性是这些酶中的一些进化出一组进化上适合的序列位置，严格地允许切换底物模糊性和手性特异性。为了探索这一假设，我们靶向突变丝氨酸酯水解酶（EH 3），其表现出令人印象深刻的71-底物库，但不是立体特异性的（e.e. 50%）。我们使用结构动作和计算进化跟踪方法来探索特异性交换序列的位置，并假设位置I244是关键的。在进化作用分析的驱动下，该位置被取代为亮氨酸，亮氨酸与异亮氨酸一起似乎是最常存在于最接近的同源序列中的氨基酸（最大值）。身份，CA。67.1%），和苯丙氨酸，出现在遥远的同系物。虽然I244 L突变没有任何功能性后果，但I244 F突变允许酯酶保持显著的53-底物范围，同时获得立体特异性性质（例如，99.99%）。这些数据支持的可能性，一些酶进化的序列位置，控制底物的范围和立体特异性。这样的残基，这可以进化筛选，可以作为起点，进一步设计底物模糊，但手性特异性，酶，在生物技术和合成化学非常赞赏。
Some enzymes evolved fitted positions controlling specificity. These positions can be evolutionary traced. Engineering substrate promiscuous yet stereospecific hydrolases is feasible. Our understanding of enzymes with high substrate ambiguity remains limited because their large active sites allow substrate docking freedom to an extent that seems incompatible with stereospecificity. One possibility is that some of these enzymes evolved a set of evolutionarily fitted sequence positions that stringently allow switching substrate ambiguity and chiral specificity. To explore this hypothesis, we targeted for mutation a serine ester hydrolase (EH3) that exhibits an impressive 71-substrate repertoire but is not stereospecific (e.e. 50%). We used structural actions and the computational evolutionary trace method to explore specificity-swapping sequence positions and hypothesized that position I244 was critical. Driven by evolutionary action analysis, this position was substituted to leucine, which together with isoleucine appears to be the amino acid most commonly present in the closest homologous sequences (max. identity, ca. 67.1%), and to phenylalanine, which appears in distant homologues. While the I244L mutation did not have any functional consequences, the I244F mutation allowed the esterase to maintain a remarkable 53-substrate range while gaining stereospecificity properties (e.e. 99.99%). These data support the possibility that some enzymes evolve sequence positions that control the substrate scope and stereospecificity. Such residues, which can be evolutionarily screened, may serve as starting points for further designing substrate-ambiguous, yet chiral-specific, enzymes that are greatly appreciated in biotechnology and synthetic chemistry.
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影响因子：
9.8
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通讯作者：
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作者：
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通讯作者：
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