Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
Molecular Enzymology Group, Groningen Institute of Biomolecular Sciences and Biotechnology, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Angew Chem Int Ed Engl. 2022 Feb 14;61(8):e202113970. doi: 10.1002/anie.202113970. Epub 2021 Dec 27.
Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric 4-oxalocrotonate tautomerase (4-OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem-fused 4-OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4-OT was then subjected to eleven rounds of directed evolution to give variant 4-OT(F11), which showed an up to 320-fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4-OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180° relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins.
基因复制和融合是产生新蛋白质的主要自然过程之一，这些新蛋白质由更简单的祖先演变而来。在这里，我们采用这种策略，将一种混杂的同六聚体 4-氧代戊烯酸互变异构酶（4-OT）进化为用于对映选择性迈克尔加成反应的高效生物催化剂。我们首先设计了串联融合的 4-OT，以允许通过定向进化对相邻亚基进行独立的序列多样化。然后，将融合的 4-OT 进行了 11 轮的定向进化，得到了变体 4-OT(F11)，它对硝基甲烷与肉桂醛的迈克尔加成反应的活性提高了 320 倍。晶体结构分析表明，4-OT(F11)具有一种不寻常的不对称三聚体结构，其中一个单体相对于其他单体翻转了 180°。这种打破结构对称性的基因复制和融合策略可能成为酶工程工具包中不可或缺的资产，在工程寡聚蛋白方面有广泛的应用。
