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.

中文翻译：

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大肠杆菌表面展示的玻璃颤菌血红蛋白的定向进化作为合成 5-亚氨基-1,2,4-噻二唑的人工金属酶

人工金属酶（ArM）是通过将有机金属催化剂锚定到可进化的蛋白质支架上而构建的。它们展现了两种组分的优点，并在体内催化新自然反应方面表现出巨大的潜力。在此，使用锚定钴卟啉辅因子而不是原始血红素辅因子的大肠杆菌表面展示的玻璃体血红蛋白（VHb ^SD-Co ）作为人工硫脲氧化酶（ATOase）来合成5-亚氨基-1,2,4-噻二唑。经过两轮组合活性位点饱和测试/迭代饱和诱变（CAST/ISM）策略的定向进化，进化出的六位点突变VHb ^SD-Co（6SM-VHb ^SD-Co）表现出催化活性的显着提高，底物范围广泛（31 个示例），全细胞产量高。这项研究展示了在新的自然反应中使用 VHb ArM 的潜力，并证明了大肠杆菌表面展示方法通过替换体内血红素蛋白中的卟啉辅因子来增强催化性能的适用性。