定向进化提高人精氨酸酶1的催化活性  

Directed evolution to enhance the catalytic activity of human arginase 1

作　　者：冯翠月 王晨宇 唐梦佳 樊帅[2] 杨兆勇[2] 张志斐 FENG Cui-yue;WANG Chen-yu;TANG Meng-jia;FAN Shuai;YANG Zhao-yong;ZHANG Zhi-fei(School of Pharmacy,North China University of Science and Technology,Tangshan 063210,China;Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050,China)

机构地区：[1]华北理工大学药学院,河北唐山063210 [2]中国医学科学院、北京协和医学院医药生物技术研究所,北京100050

出　　处：《药学学报》2024年第12期3402-3408,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金面上项目(82373767);中国医学科学院医学与健康科技创新工程(2021-I2M-1-055)

摘　　要：精氨酸酶1缺乏症(arginase 1 deficiency,ARG1-D)是一种罕见的遗传性代谢疾病,导致患者进行性痉挛性瘫痪、认知障碍和癫痫发作。来源于人源的重组人精氨酸酶1 (recombinant human arginase 1,rhArg1)是其潜在的治疗药物,但还存在活性低、半衰期短等缺陷限制了其临床应用。本研究采用定向进化的方法,通过易错PCR构建rhArg1的随机突变文库,经高通量筛选获得活性提高的突变体,联合点饱和突变探讨了R21和V182位点对活性的影响。研究发现,在反应体系中不含Mn^(2+)的条件下,突变体V182D、V182S、V182H和R21N的k_(cat)值相比于rhArg1提高2.0、1.9、1.7和1.3倍,突变体V182D、V182S、R21D和R21N的k_(cat)/K_(m)分别是rhArg1的2.1、1.7、1.4和1.4倍。突变体R21D和V182L对底物的亲和力有所增强。本研究通过定向进化和点饱和突变获得了rhArg1活性提高的突变体,从而提升了其在医疗领域的应用前景。Arginase 1 deficiency (ARG1-D) is a rare genetic metabolic disorder that leads to progressive spastic paralysis,cognitive impairment,and seizures.Recombinant human arginase 1 (rhArg1) is a potential therapeutic agent for this condition,but its clinical application is limited by low activity and short half-life.In this study,we employed directed evolution to address these issues.A random mutation library of rhArg1 was constructed using error-prone PCR,and high-throughput screening was used to identify mutants with enhanced activity.Site-saturation mutagenesis was also performed to investigate the effects of residues R21 and V182 on enzyme activity.Our findings revealed that under reaction conditions devoid of Mn^(2+),the k_(cat) values of the mutants V182D,V182S,V182H,and R21N increased by 2.0,1.9,1.7,and 1.3 times respectively,compared to rhArg1.The k_(cat)/K_(m) values of mutants V182D,V182S,R21D,and R21N were 2.1,1.7,1.4,and 1.4 times higher than those of rhArg1,respectively.Additionally,mutants R21D and V182L showed enhanced substrate affinity.Through directed evolution and site-saturation mutagenesis,we successfully obtained rhArg1 mutants with improved activity,thereby enhancing its potential for clinical application.

关 键 词：精氨酸酶1缺乏症 重组人精氨酸酶1 定向进化 饱和突变 

分 类 号：R963[医药卫生—微生物与生化药学]