FgaPT2酶催化合成C-4异戊烯基化吲哚二酮哌嗪和定向诱变增强收率  

FgaPT2 enzymatic synthesis of C-4 isopentenylated indole diketopiperazines and directed mutagenesis enhanced yield

作　　者：张弘弛 刘瑞[1,2] 高志慧 李慧[2] 杨阳[2] ZHANG Hongchi;LIU Rui;GAO Zhihui;LI hui;YANG Yang(College of Life Science,Shanxi Datong University,Datong 037009,Shanxi,China;Applied Biotechnology Institute,Shanxi Datong University,Datong 037009,Shanxi,China)

机构地区：[1]山西大同大学生命科学学院,山西大同037009 [2]山西大同大学应用生物技术研究所,山西大同037009

出　　处：《精细化工》2022年第3期633-643,共11页Fine Chemicals

基　　金：山西省应用基础研究计划(201801D221240);山西省高等学校科技创新项目(2020L0485);山西大同大学科研基金(2019K15)。

摘　　要：在二甲基烯丙基二磷酸存在下,通过FgaPT2酶催化合成了一系列C-4异戊烯基化吲哚二酮哌嗪,测试了其生物活性,对生物活性最高的产物,探讨了通过定点诱变提高酶合成的可行性。结果表明,FgaPT2酶催化合成了7个C-4异戊烯基化吲哚二酮哌嗪,FgaPT2对底物具有一定的选择性,环-L-色氨酸-L-酪氨酸(Ⅰe)异戊烯基化催化效果最好,产物环-L-4-二甲基烯丙基-色氨酸-L-酪氨酸(Ⅱe)收率达36.1%。C-4异戊烯基化显著提高了吲哚二酮哌嗪的生物活性,尤其是环-L-4-二甲基烯丙基-色氨酸-L-色氨酸(Ⅱf)对A549和MCF-7细胞达到50%抑制效果时抑制剂的浓度(IC_(50)值)分别为54.6和30.7μmol/L,对测试细菌和真菌的最低抑制浓度(MIC值)在0.5~4 mg/L,1,1-二苯基-2-三硝基苯肼自由基清除活性的IC_(50)值为98μmol/L。Arg-244的定点诱变表明,在19个突变体中,52.6%的FgaPT2突变体提高了Ⅱf收率,动力学参数验证了环-L-色氨酸-L-色氨酸(Ⅰf)与突变FgaPT2之间的相互作用,可以提高Ⅱf收率,其中R244M对Ⅰf的亲和力最高,Michaelis-Menten常数(K_(M))为0.14 mmol/L,转化数(k_(cat))为0.06471/s,k_(cat)/K_(M)为462.14 L/(s·mmol),产物收率最高,为36.9%±1.2%。In the presence of dimethylallyl diphosphate,a series of C-4 isopentenylated indole diketopiperazines were synthesized by FgaPT2 enzymatic catalysis.The bioactivity of the products was tested.For the product with the highest biological activity,the feasibility of site-directed mutagenesis to increase the yield of enzyme synthesis was studied.The results showed that seven C-4 isopentenylated indole diketopiperazines were obtained.FgaPT2 exhibited certain selectivity for substrates,and had the best catalytic effect on cyclo-L-tryptophan-L-tyrosine(Ⅰe),the product cyclo-L-4-dimethylallyl-Trp-L-Tyr(Ⅱe)had a yield of 36.1%.C-4 isopentenylation significantly improved the biological activity of indoledione piperazine.Especially,for cyclo-L-4-dimethylallyl-Trp-L-Trp(Ⅱf),the IC_(50)[half maximal(50%)inhibitory concentration]values on A549 and MCF-7 cells were 54.6 and 30.7μmol/L,respectively,the MIC(minimum inhibitory concentration)values of test bacteria and fungi were 0.5~4 mg/L,and the IC_(50) value of1,1-diphenyl-2-picrylhydrazyl radical scavenging activity was 98μmol/L.Site-directed mutagenesis of Arg-244 showed that among nineteen mutants,52.6% of FgaPT2 mutants increased the yield of Ⅱf.Kinetic parameters verified the interaction between cyclo-L-Trp-L-Trp(Ⅰf)and mutant of FgaPT2,which could increase the yield of Ⅱf.Among them,R244M had the highest affinity to Ⅰf,the Michaelis-Menten constant(KM)was 0.14 mmol/L,conversion number(k_(cat))was 0.06471/s,k_(cat)/K_(M) was 462.14 L/(s·mmol),and the product obtained the highest yield of 36.9%±1.2%.

关 键 词：吲哚二酮哌嗪 异戊二烯色氨酸合成酶 生物活性 定向诱变 精细化工中间体 

分 类 号：O629.3[理学—有机化学] TQ251.3[理学—化学]