Rational Design And Reengineering Of Allosteric Regulation Of Threonine Deaminase From Escherichia Coli

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.