摘要
异源表达鲍曼不动杆菌AB0057的UDP-葡萄糖4-差向异构酶并表征其酶学性质以及分析其结构与功能。将异构酶基因构建到pET-28a表达载体并在大肠埃希菌BL21(DE3)中异源表达,使用高效液相色谱检测酶活力及表征酶学性质。系统发育分析、序列比对、同源建模与分子对接分析其结构与关键催化位点。结果显示,重组酶Gne1获得可溶性表达,质量约为38.9 kD,催化最适温度为44℃,最适pH为6.0,米氏常数K_(M)与催化常数k_(cat)分别为(1.227±0.0824)mmol/L和(82.64±3.562)×10^(-3)·min^(-1)。该酶属于NADB_Rossmann超家族并分属于UDP_G4E_1_SDR_e亚组,分别具有典型GXGXXG基序与YXXXK基序。N端结构域与NAD结合,而C端结构域用来结合底物,催化关键位点为S125和Y150。本研究验证了Gne1的差向异构酶活性,阐释了其序列特点和结构特征,揭示了其与底物、辅因子的结合模式,分析了关键催化位点。为蛋白质工程改造提高酶活力进而利用生物酶法合成稀有功能糖提供了理论依据。
The purpose of this article is to express the UDP-glucose 4-epimerase from Acinetobacter baumannii AB0057,characterize its enzymatic properties and analyze its structure and function.The epimerase gene was constructed into pET-28a expression vector and heterologously expressed in BL21(DE3).Enzyme activity was assayed using high performance liquid chromatography.The structure and key residues were analyzed by phylogenetic analysis,sequence alignment,homology modeling and molecular docking.Results indicated that the recombinant enzyme Gne1 was expressed at a molecular weight of 38.9 kD,with an optimum temperature of 44℃ and an optimum pH of 6.0.Michaelis-Menten parameters K_(M) and k_(cat) were(1.227±0.0824)mmol/L and(82.64±3.562)×10^(-3)·min^(-1),respectively.This enzyme belongs to NADB_Rossmann superfamily and UDP_G4E_1_SDR_e subgroup with typical GXGXXG and YXXXK sequence motifs.The N-terminal structural domain bound to NAD,while the C-terminal structural domain bound to substrate,and the catalytic key sites were S125 and Y150.The current work verified the epimerase activity of Gne1,explained its sequence and structural features,revealed its binding mode with substrates and cofactors,and analyzed the key residues,which provides a basis for protein engineering to improve the epimerase activity and then use biological enzymatic method to synthesize rare functional sugars.
作者
张展
冯雁
李谦
崔莉
ZHANG Zhan;FENG Yan;LI Qian;CUI Li(State Key Laboratory of Microbial Metabolism,School of Life Science and Technology,Shanghai Jiao Tong University,Shanghai 200240;School of Life Science&Technology,China Pharmaceutical University,Nanjing 210009,China)
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2021年第6期742-750,共9页
Journal of China Pharmaceutical University
基金
国家自然科学基金资助项目(No.21977067,No.31770098,No.31620103901)
国家重点研发计划项目资助(2018YFA0900702)。
关键词
差向异构酶
酶学性质表征
序列及结构分析
稀有功能糖合成
epimerase
enzymatic property
sequence and structure analysis
synthesis of rare functional sugar
