Dongping Chen, Xiang Zhang, Anastassia Andreevna Vorobieva, Ryo Tachibana, Alina Stein, Roman P. Jakob, Zhi Zou, Damian Alexander Graf, Ang Li, Timm Maier, Bruno E. Correia, Thomas R. Ward
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引用次数: 0
Abstract
While natural terpenoid cyclases generate complex terpenoid structures via cationic mechanisms, alternative radical cyclization pathways are underexplored. The metal-catalysed H-atom transfer reaction (M-HAT) offers an attractive means for hydrofunctionalizing olefins, providing access to terpenoid-like structures. Artificial metalloenzymes offer a promising strategy for introducing M-HAT reactivity into a protein scaffold. Here we report our efforts towards engineering an artificial radical cyclase (ARCase), resulting from anchoring a biotinylated [Co(Schiff-base)] cofactor within an engineered chimeric streptavidin. After two rounds of directed evolution, a double mutant catalyses a radical cyclization to afford bicyclic products with a cis-5-6-fused ring structure and up to 97% enantiomeric excess. The involvement of a histidine ligation to the Co cofactor is confirmed by crystallography. A time course experiment reveals a cascade reaction catalysed by the ARCase, combining a radical cyclization with a conjugate reduction. The ARCase exhibits tolerance towards variations in the dienone substrate, highlighting its potential to access terpenoid scaffolds.
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