摘要
精氨酸脱亚胺酶(arginine deiminase,EC 3.5.3.6,ADI)因其可作为精氨酸营养缺陷型肿瘤细胞的靶向治疗药物而受到广泛关注.目前,支原体来源的重组ADI处于肝癌和黑素瘤的三期临床研究阶段.作为药用酶,当前报道的ADI在体内生理条件下普遍存在酶活低、半衰期短、底物亲和性弱等局限性.本研究结合随机突变及基于理性设计的定点突变两种方法,对研究室前期自主筛选得到的变形假单胞菌Pseudomonas plecoglossicida来源的ADI经一轮定向进化后所获优势突变株M314(A128T/H404R/I410L)进行分子改造.通过对随机突变法获得的1480个突变株进行96孔板高通量筛选,得到优良突变株M173(A128T/H404R/I410L/K272R);同时,基于同源序列比对及ADI蛋白三维结构同源建模,采用PyMOL软件理性预测和分析其活性中心及附近保守区域氨基酸位点对蛋白功能的影响,选择了6个位点D78E、L223I、P230I、S245D、A275N、R400M分别在M314的基础上进行定点突变,最终获得优势突变株M04(A128T/H404R/I410L/S245D).通过对突变株的酶学性质以及动力学参数分析发现:生理pH值下,突变株M173的酶比活(12.32 U/mg)在M314(9.02 U/mg)的基础上提升36.59%,Kcat/Km提高52.36%;而突变株M04的最适pH由6.5升高至7.0,更接近体内生理pH,其比酶活(14.66 U/mg)较M314提升62.53%,Kcat/Km提高了37.12%.综上结果,本研究结合两种分子改造方法成功地对该ADI在生理pH条件下的酶活和酶学性质进行了改良,并为蛋白质的分子改造策略提供了理论基础和实验依据.
Arginine deiminase （ADI, EC 3.5.3.6） was recently recognized as a drug target forarginine-auxotrophic tumors. Recombinant ADI from Mycoplasma arginini is currently being tested for the treatment of hepatocellular carcinomas （HCCs） and melanomas in phase Ⅲ trials. The demanded improvement microbial ADIs includes to increase the substrate affinity, specific activity, and half-lifeunder physiological pH conditions. In this study, we combined random mutagenesis and site-directed mutagenesis based on a rational design for M314 （A128T/H404R/I410L）. ADI mutant from Pseudomonas plecoglossicida was previously described. After an additional round of ep-PCR and HTS （high throughput screening） in 96-well plate, an mutant M173 （A128T/H404R/I410L/K272R） was isolated from 1480 mutants. According to the 3D structure of ADI, six point mutations on M314 including M01-D78E, M02-L223I, M03-P230I, M04-S245D, M05-A275N, and M06-R400M were recognized as the several key residues at active center and nearby conserved domain. By QuikChange site-directed mutagenesis, the mutant M04 （A128T/H404R/I410L/S245D） was selected for further analysis. The results showed that the specific activity and Kcat/Km of M173 were increased by 36.59% （ 12.32 U/mg） and 52.36% compared with M314 （9.02 U/mg）. The optimum pH of mutant M04 was shifted from 6.5 to 7.0 with specific activity and Kcat/Km values of 62.53% （14. 66 U/mg） and the increase of 37. 12%, than M314. In summary, the enzymatic properties of our ADI mutant at physiological pH were improved from the design of theoretical and experimental rationales.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2013年第2期175-182,共8页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金(No.30900030)
教育部新世纪优秀人才支持计划(No.NCET-11-0658)
国家重点基础研究发展计划(973计划
No.2011CB710800)
111引智计划(No.111-2-06)
江苏高校优势学科建设工程资助项目~~
关键词
精氨酸脱亚胺酶
随机突变
定点突变
三维结构分析
arginine deiminase
random mutagenesis
site-directed mutagenesis
3D structure simulating
