Biocatalysts are sought-after in synthesis of pharmaceuticals and agrochemicals due to their high regioselectivity and enantioselectivity. Among biocatalysts, heme-containing cytochrome P450 (P450) oxygenases are an attractive target since they catalyze oxidation of “unactivated” carbon-hydrogen bonds with high efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius, which has the potential to be widely used as a biocatalyst since it shows activity at high temperatures and low pH. Polyhistidine tags (His-tags) are widely used to simplify purification of proteins. However, His-tags can cause changes to protein structure and function. Here, we demonstrate the effects of His-tags on CYP119. To this end, the His-tags were cloned at the N-terminus or C-terminus of the CYP119, and His-tagged proteins were expressed and isolated. The thermostability and peroxidase activity of His-tagged CYP119s were tested and compared to wild type CYP119. Results indicated that while addition of His-tags increased the yield and simplified isolation of CYP119, they also influenced the electronic structure of active site and the activity of the protein. We show that N-terminal His-tagged CYP119 has desirable properties and potential to be used in industrial applications, but mechanistic studies using this protein need careful interpretation since the His-tag affects electronic properties of the active site heme iron.

中文翻译：

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N末端和C末端多组氨酸标签对嗜热性P450 CYP119稳定性和功能的影响。

由于其高的区域选择性和对映选择性，生物催化剂在药物和农用化学品的合成中很受欢迎。在生物催化剂中，含血红素的细胞色素P450（P450）加氧酶是诱人的目标，因为它们能高效催化“未激活的”碳氢键的氧化。CYP119是来自Sulfolobus acidocaldarius的耐酸P450由于其在高温和低pH下具有活性，因此有可能被广泛用作生物催化剂。聚组氨酸标签（His标签）被广泛用于简化蛋白质的纯化。但是，His标签会引起蛋白质结构和功能的改变。在这里，我们证明了His标签对CYP119的影响。为此，将His标签克隆到CYP119的N末端或C末端，并表达和分离His标签的蛋白。测试了带有His标签的CYP119s的热稳定性和过氧化物酶活性，并将其与野生型CYP119进行了比较。结果表明，虽然添加His-tags可以提高CYP119的产量并简化其分离，但它们还影响了活性位点的电子结构和蛋白质的活性。