定点突变解析短乳杆菌酪氨酸脱羧酶的关键催化位点  

Mapping of key catalytic residues of Lactobacillus brevis L⁃tyrosine decarboxylase using site⁃directed mutagenesis

作　　者：倪婕 许国超[1] 倪晔[1] NI Jie;XU Guochao;NI Ye(Key Laboratory of Industrial Biotechnology of the Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《生物加工过程》2021年第1期32-39,共8页Chinese Journal of Bioprocess Engineering

基　　金：国家轻工技术与工程一流学科计划(LITE201807);国家自然科学基金(21776112)。

摘　　要：酪氨酸脱羧酶(TDC)是一种磷酸吡哆醛(PLP)依赖型的氨基酸脱羧酶,可用于催化L酪氨酸脱羧生成酪胺。酪胺是一种重要的生物单胺,在生物医药及化工产业中具有广泛应用价值。以Lactobacillus brevis来源的TDC晶体结构为基础,通过分子对接和作用力分析得到了参与底物识别及结合的关键区域和位点,包括H98~E102的loop区域以及Y331和Y398位点,并对这些位点进行定点突变。对突变体进行蛋白纯化和比酶活分析,与野生酶相比,突变体S101A、E102D对L酪氨酸分别保留66.9%及69.1%的相对活力,对L多巴(L-DOPA)分别保留74.2%及61.3%的相对活力,其余突变体残余活力均低于10%。通过分子动力学模拟分析发现,H98与N100分别与底物的羧基和酚羟基形成氢键,Y331的苯环与底物的苯环形成ππ堆积作用。H98~E102的loop区域与Y331和Y398共同识别底物并固定底物苯环与PLP的共轭π平面相垂直,促进脱羧反应的高效发生。Tyrosine decarboxylase(TDC) is a pyridoxal phosphate(PLP) dependent amino acid decarboxylase.It catalyzes the decarboxylation of L-tyrosine into tyramine,an important biogenic monoamine.Tyramine is widely used in pharmaceutical and chemical industry.Based on the crystal structure of Lactobacillus brevis tyrosine decarboxylase(LbTDC),molecular docking was done to analyze the key residues involved in substrate binding and catalysis.Site-directed mutagenesis was performed in H98-E102 loop region,residues Y331 and Y398 to investigate their potential contribution to decarboxylation.All the variants were purified and onlyvariants S101 A and E102 D retained approximately 66.9% and 69.1% of the activity of wild type LbTDC towards L-tyrosine,74.2% and 61.3% of the activity of the wild type towards L-DOPA,respectively,whereas other mutants were almost inactivated.Molecular dynamic analysis indicates that H98 and N100 form hydrogen bonds with L-tyrosine,and benzene ring of Y331 interacts with benzene group of L-tyrosine through π-π stacking interaction.The H98-E102 loop region,together with Y331 and Y398 playes important roles in recognizing the substrate and anchoring the benzene ring of L-tyrosine perpendicular to the conjugated π system of PLP,to promote the efficient decarboxylation reaction.

关 键 词：短乳杆菌 酪氨酸脱羧酶 L酪氨酸 定点突变 分子动力学模拟 底物结合 酪胺 

分 类 号：Q556[生物学—生物化学]