谷氨酸棒杆菌天冬氨酸激酶G359D突变解除赖氨酸与苏氨酸协同抑制的研究  

Aspartokinase G359D from Corynebacterium glutamicum Relieves the Synergistic Inhibition of Lysine and Threonine

作　　者：徐德雨 郑小梅[2] 赵晶[2] 郑平[2] 赵树欣[1] 

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院系统微生物工程重点实验室,天津300308

出　　处：《生物技术通报》2017年第11期143-152,共10页Biotechnology Bulletin

基　　金：中国科学院重点部署项目(ZDRW-ZS-2016-2)

摘　　要：天冬氨酸激酶(Aspartate Kinase,AK)是赖氨酸合成途径中关键酶,其受到代谢产物赖氨酸与苏氨酸的协同抑制,旨在发现其解除协同抑制的新突变体并解析其机理。通过对赖氨酸高产菌Corynebacterium glutamicum ZL5与野生型C.glutamicumATCC 13032的天冬氨酸激酶氨基酸序列比对,发现在高产菌的天冬氨酸激酶中存在G359D突变。通过体外天冬氨酸激酶野生型和G359D突变体酶活检测,发现该G359D突变体在10 mmol/L赖氨酸和苏氨酸同时存在时仍保留了76.94%±1.61%的酶活,而野生酶的酶活仅残留4.38%±1.28%。这一结果在赖氨酸高产菌谷氨酸棒杆菌的回复突变中也可得到验证,其回复突变后使赖氨酸产量下降15.57%。通过同源建模和结构分析发现,与野生酶相比,G359D突变体结合赖氨酸后,其位于活性中心附近的Arg151与Glu74之间无法形成离子键,允许底物分子的结合而具有较高活性。发现天冬氨酸激酶G359D突变体可阻断赖氨酸引起的别构效应,从而有效解除赖氨酸与苏氨酸的协同抑制。Aspartokinase（AK）is a key enzyme involved in the lysine biosynthesis,but its activity is synergistically inhibited byend-products such as lysine and threonine. This study focuses on finding new mutation to relieve the synergistic inhibition and unveiling thismolecular mechanism form protein structure analysis. The amino acid sequences of AK from high lysine-producing strain Corynebacterium glutamicum ZL5 and wild-type（WT）C. glutamicum ATCC 13032 were aligned,and it was shown that G359 D mutation was detected inaspartokinase from C. glutamicum ZL5. To discovery the biological function of this mutation,these WT and G359 D Aks were expressed inEscherichia coli and were purified by the affinity chromatography;then,the purified recombined proteins were used for enzymatic activitydetection when added the inhibitors lysine and threonine. The G359 D protein displayed high resistance against the synergistic inhibition oflysine and threonine. The enzymatic activity of G359 D remained at 76.94%±1.61% when the concentration of lysine and threonine reached 10 mmol/L,but the WT protein only was in 4.38%±1.28%. The similar result was also verified by the reverse mutation of G359 D in the genomeof C. glutamicum ZL5 producing high lysine yield,and the lysine yield decreased by 15.57%. From the further homology modeling and proteinstructure analysis,the G359 D still interacted with lysine and threonine,but it enabled to relieve the allosteric effect of lysine,resulting fromthat the Arg151 and Glu74 in the catalytic active site of G359 D could not form the ionic bond,thus allow the substrate into the active site. Theaspartokinases G359 D enabled to relieve the synergistic inhibition of lysine and threonine by blocking the allosteric effect of lysine.

关 键 词：谷氨酸棒杆菌 天冬氨酸激酶 赖氨酸 苏氨酸 协同抑制 

分 类 号：Q55[生物学—生物化学]