【作者】 陈林；

【导师】 曾安平；

【作者基本信息】 天津大学 ， 生物化工， 2012， 硕士

【摘要】 生物合成型苏氨酸脱氨酶（TD）是异亮氨酸（Ile）生物合成途径中的关键酶，受终端产物Ile的反馈抑制及竞争途径产物缬氨酸（Val）的激活。解除Ile对TD的反馈抑制对Ile生产至关重要，但是Ile和Val在TD的结合位点尚不清楚，而这对理性设计TD构建非Ile敏感性突变体是非常必要的。基于大肠杆菌磷酸甘油酸脱氢酶、拟南芥的天冬氨酸激酶1及大肠杆菌TD的ACT-domain结构比对结果，准确预测了Ile和Val在TD上可能的结合位点。根据氨基酸的保守性选择了其中的14个氨基酸进行定点突变，并对这些突变体进行了Ile及Val敏感性分析和酶动力学性质研究。研究结果表明每一个TD单体都有两个非等价的效应物结合位点：位点A包括残基R362、E442、G445、A446、R466和H468；位点B包括残基E347、G350、F352、Y369、I460和S461，都与Ile相互作用，其中E347、G350和F352也参与结合Val。结合分子对接结果，本论文中提议了一种基于变构效应物Ile和Val浓度控制的调控机制。根据结合位点的研究结果，理性设计并构建出的5个TD双位点突变体对Ile敏感性显著减弱，证明了结合位点的研究对于理性设计Ile非敏感型TD突变体具有重要的指导意义。其中，TD_R362F/I460F相对于野生型TD对Ile的抗性至少提高了700倍，甚至完全解除了Ile的反馈抑制，且无效应物时两者的酶活水平相当；TD_F352A/R362F不仅酶活水平提高，对底物亲和力更强，而且对Ile的敏感性减弱非常显著。在大肠杆菌JW3591中过表达该突变体比过表达野生型TD或TdcB能显著增加Ile和2-酮丁酸的生产。从酶学的角度讲，TD突变体TD_R362F/I460F和TD_F352A/R362F对开发Ile或其他以2-酮丁酸为前体的化合物的高产菌株具有极高的应用价值。为了探讨TD调控域作为调控元件用于蛋白质调控性能改造的可行性，我们将TD的调控域移植到与TD催化域结构高度相似的生物降解型苏氨酸脱氨酶（TdcB）上构建融合蛋白TdcBR，同时移除TD的调控域构建突变体TDdR。Ile敏感性分析表明TdcB和TDdR对Ile不敏感，而TdcBR1对Ile有较弱的敏感性，证明TD调控域作为调控元件可用于蛋白质工程化设计。更多还原

【Abstract】 Biosynthetic threonine deaminase （TD） is a key enzyme for the synthesis ofisoleucine which is allosterically inhibited and activated by isoleucine （Ile） and valine（Val）, respectively. The binding sites of Ile and Val and the mechanism of theirregulations in TD are not clear, but essential for a rational design of efficientproductive strain（s） for Ile and related amino acids.The potential binding sites of the allosteric effectors were accurately predictedbased on structural comparison among ACT domains of TD and PGDH（phosphoglycerate dehydrogenase） from E. coli and AK1（aspartokinase1） fromArabidopsis thaliana. Then14TD mutants were created by site-directed mutagenesisaccording to the conservation. Sensitivity analysis of these mutants to Ile and Val andthe enzyme kinetic studies reveal that each regulatory domain of the TD monomerpossesses two nonequivalent effector-binding sites, site A and site B. The residuesR362, E442, G445, A446, R466and H468belonging to site A only interact with Ile,while E347, G350, F352, Y369, I460and S461from site B are not only involved inthe Ile binding but the residues E347、G350and F352also in the Val binding. Byfurther considering enzyme kinetic data and the docking results aconcentration-dependent mechanism of the allosteric regulation of TD by Ile and Valwas proposed in this study.Based on the study of binding sites, five more feedback-insensitive mutants withdouble mutantion were designed and created. It proved that the study of the bindingsite valuably guides us to reengineer Ile insensitive mutants. Among them, the mutantTD_R362F/I460Fis more than700-fold insensitive to Ile as compared to the wild-type TDor even completely insensitive to Ile anymore, but has the same activity level as thewild-type TD without any effectors. The mutant TD_F352A/R362Fshowed both higheractivity and substrate affinity and much stronger resistance to Ile inhibition comparedto those of wildtype enzyme. Overexpression of this mutant in E. coli JW3591significantly increased the production of ketobutyrate and Ile in comparison to thereference strains overexpressing wildtype TD or the catabolic threonine deaminase（TdcB）. In view of enzymology, the mutants TD_R362F/I460Fand TD_F352A/R362Farevalueable for developing strains which can overproduce Ile and other chemical using2-ketobutyrate as a precursor. To test the feasibility that the regulatory domain of TD treating as a regulatoryelement to alter protein regulation performance. We fused the regulatory domain ofTD with the biodegradative threonine deaminase （TdcB） which shares high structuralconservation with the TD catalytic donmain to generate mutant TdcBR and removedthe regulatory domain of TD to create TDdR. Ile sensitivity analysis show that TDdRand TdcB are Ile-insensitive while TdcBR1is weakly sensitive to Ile. It suggested thatthe regulatory domain of TD is potential to be a regulatory element for reengineeringprotein.更多还原