靛蓝生产确定了细胞色素 P450 BM3 中芳香烃羟化多样化的热点。

IF 3.4 3区 化学 Q2 Chemistry
Douglas J. Fansher, Jonathan N. Besna and Joelle N. Pelletier
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引用次数: 0

摘要

P450 BM3 的进化是一个广泛的研究课题,但筛选各种底物/反应组合仍然是一个耗时的过程。靛蓝的产生有可能成为一种简单的高通量反应筛选方法,因为表达靛蓝(+)变体的细菌菌落可以通过其蓝色表型直观地识别出来。使用 4-氨基安替比林比色法筛选了靛蓝(+)单一变体、靛蓝(-)单一变体和组合库(其中包含可产生蓝色表型的突变)羟化 12 种芳香族化合物的能力。重组靛蓝(+)单变体以创建多变体库是一种特别有用的策略,因为所有具有高羟化活性的 P450 BM3 顶级变体要么是靛蓝(+)单变体,要么包含多个取代。此外,利用 4-AAP 分析法确定的活性变体得到了进一步表征,并确定了几个变体,这些变体与 1,3-二氯苯的转化率超过 90%,并主要形成 2,6-二氯苯酚;其他变体则表现出明显的底物选择性。这支持了一种假设,即在能够产生靛蓝(+)表型的位置或热点残基上进行取代是提高芳香烃羟基化活性的一般机制。总之,这项研究表明,通过比色菌落筛选鉴定出的靛蓝(+)单一变体可以通过重组产生一个多取代变体库,其中包含许多具有高芳香羟化活性的变体。基于菌落的筛选与其他筛选测定法的结合大大加快了酶工程的进程,因为无需对单一变体进行广泛筛选,就可以将容易识别的靛蓝(+)单一变体进行重组,生成具有活性的多重变体库。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Indigo production identifies hotspots in cytochrome P450 BM3 for diversifying aromatic hydroxylation†

Indigo production identifies hotspots in cytochrome P450 BM3 for diversifying aromatic hydroxylation†

Indigo production identifies hotspots in cytochrome P450 BM3 for diversifying aromatic hydroxylation†

Evolution of P450 BM3 is a topic of extensive research, but screening the various substrate/reaction combinations remains a time-consuming process. Indigo production has the potential to serve as a simple high-throughput method for reaction screening, as bacterial colonies expressing indigo (+) variants can be visually identified via their blue phenotype. Indigo (+) single variants, indigo (−) single variants and a combinatorial library, containing mutations that enable the blue phenotype, were screened for their ability to hydroxylate a panel of 12 aromatic compounds using the 4-aminoantipyrine colorimetric assay. Recombination of indigo (+) single variants to create a multiple-variant library is a particularly useful strategy, as all top performing P450 BM3 variants with high hydroxylation activity were either indigo (+) single variants or contained multiple substitutions. Furthermore, active variants, as determined using the 4-AAP assay, were further characterized and several variants were identified that gave more than 90% conversion with 1,3-dichlorobenzene and predominantly formed 2,6-dichlorophenol; other variants showed significant substrate selectivity. This supports the hypothesis that substitution at positions that enable the indigo (+) phenotype, or hotspot residues, is a general mechanism for increasing aromatic hydroxylation activity. Overall, this research demonstrates that indigo (+) single variants, identified via colorimetric colony-based screening, may be recombined to generate a multiply-substituted variant library containing many variants with high aromatic hydroxylation activity. The combination of colony-based screening and other screening assays greatly accelerates enzyme engineering, as readily-identified indigo (+) single variants can be recombined to create a library of active multiple variants without extensive screening of single variants.

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来源期刊
Faraday Discussions
Faraday Discussions CHEMISTRY, PHYSICAL-
CiteScore
4.90
自引率
0.00%
发文量
259
审稿时长
2.8 months
期刊介绍: Discussion summary and research papers from discussion meetings that focus on rapidly developing areas of physical chemistry and its interfaces
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