Directed evolution of surface-displayed hemoglobin as an artificial metalloenzyme for the synthesis of 5-imino-1,2,4-thiadiazoles

• Published in: Chemical science (Cambridge) Vol. 15; no. 2; pp. 7742 - 7748
• Main Authors: Xu, Yaning, Li, Fengxi, Xie, Hanqing, Liu, Yuyang, Han, Weiwei, Wu, Junhao, Cheng, Lei, Wang, Chunyu, Li, Zhengqiang, Wang, Lei
• Format: Journal Article
• Published: 22.05.2024
• ISSN: 2041-6520; 2041-6539
• DOI: 10.1039/d4sc00005f

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 (VHb SD-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 VHb SD-Co (6SM-VHb SD-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 . Escherichia coli surface-displayed Vitreoscilla hemoglobin as an artificial metalloenzyme for the synthesis of 5-imino-1,2,4-thiadiazoles.