Aslantas Yaprak, Surmeli Nur Basak
Program in Biotechnology and Bioengineering, İzmir Institute of Technology, İzmir, Turkey.
Department of Bioengineering, İzmir Institute of Technology, İzmir, Turkey.
Bioinorg Chem Appl. 2019 Jun 20;2019:8080697. doi: 10.1155/2019/8080697. eCollection 2019.
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 , 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.
由于生物催化剂具有高区域选择性和对映选择性，因此在药物和农用化学品的合成中备受青睐。在生物催化剂中，含血红素的细胞色素P450（P450）加氧酶是一个有吸引力的目标，因为它们能高效催化“未活化”碳氢键的氧化。CYP119是一种来自[具体来源未提及]的嗜酸嗜热P450，由于它在高温和低pH条件下仍具有活性，因此有潜力被广泛用作生物催化剂。多组氨酸标签（His标签）被广泛用于简化蛋白质的纯化。然而，His标签可能会导致蛋白质结构和功能的改变。在此，我们展示了His标签对CYP119的影响。为此，将His标签克隆到CYP119的N端或C端，表达并分离出带His标签的蛋白质。测试了带His标签的CYP119的热稳定性和过氧化物酶活性，并与野生型CYP119进行比较。结果表明，虽然添加His标签提高了CYP119的产量并简化了其分离，但它们也影响了活性位点的电子结构和蛋白质的活性。我们表明，N端带His标签的CYP119具有理想的特性和在工业应用中的潜力，但使用该蛋白质进行的机理研究需要仔细解读，因为His标签会影响活性位点血红素铁的电子性质。
