Li Lifeng, Ni Ye, Sun Zhihao
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2012 Apr;28(4):508-19.
Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.
精氨酸脱亚氨酶（ADI）作为一种潜在的抗癌药物，已在III期临床试验中用于抑制精氨酸营养缺陷型肿瘤（如黑色素瘤和肝细胞癌）。在这项工作中，我们通过定点诱变研究了精氨酸脱亚氨酶活性的分子机制。利用QuikChange定点诱变方法将三个突变位点A128、H404和I410引入野生型ADI基因，获得了四个ADI突变体M1（A128T）、M2（H404R）、M3（I410L）和M4（A128T，H404R）。将ADI突变体分别在大肠杆菌BL21（DE3）中表达，并测定纯化的突变蛋白的酶学性质。结果表明，A128T和H404R在生理条件（pH 7.4）下均提高了ADI的最适pH、活性和稳定性，同时降低了K(m)值。本研究为ADI活性的分子机制提供了深入了解，也为未来合理的蛋白质进化提供了实验依据。
