Directed evolution of Escherichia coli surface-displayed Vitreoscilla hemoglobin as an artificial metalloenzyme for the synthesis of 5-imino-1,2,4-thiadiazoles†
Abstract
Artificial metalloenzymes (ArMs) are constructed by anchoring organometallic catalysts to an evolvable protein scaffold. They present the advantages of both components and exhibit considerable potential for the in vivo catalysis of new-to-nature reactions. Herein, Escherichia coli surface-displayed Vitreoscilla hemoglobin (VHbSD-Co) that anchored the cobalt porphyrin cofactor instead of the original heme cofactor was used as an artificial thiourea oxidase (ATOase) to synthesize 5-imino-1,2,4-thiadiazoles. After two rounds of directed evolution using combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy, the evolved six-site mutation VHbSD-Co (6SM-VHbSD-Co) exhibited significant improvement in catalytic activity, with a broad substrate scope (31 examples) and high yields with whole cells. This study shows the potential of using VHb ArMs in new-to-nature reactions and demonstrates the applicability of E. coli surface-displayed methods to enhance catalytic properties through the substitution of porphyrin cofactors in hemoproteins in vivo.
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