Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel–Crafts Alkylase,ACS Catalysis

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Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel–Crafts Alkylase
ACS Catalysis ( IF 11.3 ) Pub Date : 2021-05-26 , DOI: 10.1021/acscatal.1c00996
Reuben B Leveson-Gower ₁ , Zhi Zhou ₁ , Ivana Drienovská ₁ , Gerard Roelfes ₁

Affiliation

The construction and engineering of artificial enzymes consisting of abiological catalytic moieties incorporated into protein scaffolds is a promising strategy to realize non-natural mechanisms in biocatalysis. Here, we show that incorporation of the noncanonical amino acid para-aminophenylalanine (pAF) into the nonenzymatic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel–Crafts alkylation between α,β-unsaturated aldehydes and indoles. pAF acts as a catalytic residue, activating enal substrates toward conjugate addition via the formation of intermediate iminium ion species, while the protein scaffold provides rate acceleration and stereoinduction. Improved LmrR_pAF variants were identified by low-throughput directed evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of aliphatic enals and substituted indoles. Analysis of Michaelis–Menten kinetics of LmrR_pAF and evolved mutants reveals that different activities emerge via evolutionary pathways that diverge from one another and specialize catalytic reactivity. Translating this iminium-based catalytic mechanism into an enzymatic context will enable many more biocatalytic transformations inspired by organocatalysis.

中文翻译：

解锁人工酶中的亚胺催化作用以创建弗里德尔-克来福特烷基化酶

由掺入蛋白质支架的非生物催化部分组成的人工酶的构建和工程是实现生物催化非自然机制的有前途的策略。在这里，我们证明，将非经典氨基酸对氨基苯丙氨酸（pAF）掺入非酶蛋白支架LmrR中，可以产生一种高效且立体选择性的人工酶（LmrR_pAF），用于α,β-不饱和醛和吲哚之间的插烯弗里德尔-克来福特烷基化。 pAF 充当催化残基，通过形成中间亚胺离子种类来激活烯醇底物以进行缀合物加成，而蛋白质支架则提供速率加速和立体诱导。通过丙氨酸扫描建议的低通量定向进化鉴定了改进的 LmrR_pAF 变体，以获得三重突变体，该突变体为一系列脂肪族烯醛和取代的吲哚提供更高的产量和对映选择性。对 LmrR_pAF 和进化突变体的 Michaelis-Menten 动力学分析表明，不同的活性通过彼此不同且专门化催化反应性的进化途径出现。将这种基于亚胺的催化机制转化为酶促环境将使更多受有机催化启发的生物催化转化成为可能。

更新日期：2021-06-18

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