Molecular insights into the regulatory landscape of PKC-related kinase-2 (PRK2/PKN2) using targeted small compounds.

The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Allosteric regulation of kinase activity.

The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.

Nucleotide binding to the ATP-cone in anaerobic ribonucleotide reductases allosterically regulates activity by modulating substrate binding.

A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.

Unleashing the Power of Evolution in Xylanase Engineering: Investigating the Role of Distal Mutation Regulation.

The drive to enhance enzyme performance in industrial applications frequently clashes with the practical limitations of exhaustive experimental screening, underscoring the urgency for more refined and strategic methodologies in enzyme engineering. In this study, xylanase Xyl-1 was used as the model, coupling evolutionary insights with energy functions to obtain theoretical potential mutants, which were subsequently validated experimentally. We observed that mutations in the nonloop region primarily aimed at enhancing stability and also encountered selective pressure for activity. Notably, mutations in this region simultaneously boosted the Xyl-1 stability and activity, achieving a 65% success rate. Using a greedy strategy, mutant M4 was developed, achieving a 12 °C higher melting temperature and doubled activity. By integration of spectroscopy, crystallography, and quantum mechanics/molecular mechanics molecular dynamics, the mechanism behind the enhanced thermal stability of M4 was elucidated. It was determined that the activity differences between M4 and the wild type were primarily driven by dynamic factors influenced by distal mutations. In conclusion, the study emphasizes the pivotal role of evolution-based approaches in augmenting the stability and activity of the enzymes. It sheds light on the unique adaptive mechanisms employed by various structural regions of proteins and expands our understanding of the intricate relationship between distant mutations and enzyme dynamics.

An RNA aptamer exploits exosite-dependent allostery to achieve specific inhibition of coagulation factor IXa.

Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.

Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase.

Regulatory cystathionine ß-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.

G Protein-Coupled Receptors: A Century of Research and Discovery.

GPCRs (G protein-coupled receptors), also known as 7 transmembrane domain receptors, are the largest receptor family in the human genome, with ≈800 members. GPCRs regulate nearly every aspect of human physiology and disease, thus serving as important drug targets in cardiovascular disease. Sharing a conserved structure comprised of 7 transmembrane α-helices, GPCRs couple to heterotrimeric G-proteins, GPCR kinases, and ß-arrestins, promoting downstream signaling through second messengers and other intracellular signaling pathways. GPCR drug development has led to important cardiovascular therapies, such as antagonists of ß-adrenergic and angiotensin II receptors for heart failure and hypertension, and agonists of the glucagon-like peptide-1 receptor for reducing adverse cardiovascular events and other emerging indications. There continues to be a major interest in GPCR drug development in cardiovascular and cardiometabolic disease, driven by advances in GPCR mechanistic studies and structure-based drug design. This review recounts the rich history of GPCR research, including the current state of clinically used GPCR drugs, and highlights newly discovered aspects of GPCR biology and promising directions for future investigation. As additional mechanisms for regulating GPCR signaling are uncovered, new strategies for targeting these ubiquitous receptors hold tremendous promise for the field of cardiovascular medicine.

Characterization of chloroplastic thioredoxin dependent glutathione peroxidase like protein in Euglena gracilis: biochemical and functional perspectives.

Euglena gracilis, a fascinating organism in the scientific realm, exhibits characteristics of both animals and plants. It maintains redox homeostasis through a variety of enzymatic and non-enzymatic antioxidant molecules. In contrast to mammals, Euglena possesses nonselenocysteine glutathione peroxidase homologues that regulate its intracellular pools of reactive oxygen species. In the present study, a full-length cDNA of chloroplastic EgGPXL-1 was isolated and subjected to biochemical and functional characterization. Recombinant EgGPXL-1 scavenged H2O2 and t-BOOH, utilizing thioredoxin as an electron donor rather than glutathione. Despite its monomeric nature, EgGPXL-1 exhibits allosteric behavior with H2O2 as the electron acceptor and follows typical Michaelis-Menten kinetics with t-BOOH. Suppression of EgGPXL-1 gene expression under normal and high-light conditions did not induce critical situations in E. gracilis, suggesting the involvement of compensatory mechanisms in restoring normal conditions.

Structural perspectives on chemokine receptors.

Chemokine receptors are integral to the immune system and prime targets in drug discovery that have undergone extensive structural elucidation in recent years. We outline a timeline of these structural achievements, discuss the intracellular negative allosteric modulation of chemokine receptors, analyze the mechanisms of orthosteric receptor activation, and report on the emerging concept of biased signaling. Additionally, we highlight differences of G-protein binding among chemokine receptors. Intracellular allosteric modulators in chemokine receptors interact with a conserved motif within transmembrane helix 7 and helix 8 and exhibit a two-fold inactivation mechanism that can be harnessed for drug-discovery efforts. Chemokine recognition is a multi-step process traditionally explained by a two-site model within chemokine recognition site 1 (CRS1) and CRS2. Recent structural studies have extended our understanding of this complex mechanism with the identification of CRS1.5 and CRS3. CRS3 is implicated in determining ligand specificity and surrounds the chemokine by almost 180°. Within CRS3 we identified the extracellular loop 2 residue 45.51 as a key interaction mediator for chemokine binding. Y2917.43 on the other hand was shown in CCR1 to be a key determinant of signaling bias which, along with specific chemokine-dependent phosphorylation ensembles at the G-protein coupled receptors (GPCR's) C-terminus, seems to play a pivotal role in determining the direction of signal bias in GPCRs.

A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling.

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize a phosphorylation site at Ser325 situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.

Exploring the molecular mechanisms of Lrp/AsnC-type transcription regulator DecR, an L-cysteine-responsive feast/famine regulatory protein.

The Lrp/AsnC family of transcriptional regulators is commonly found in prokaryotes and is associated with the regulation of amino acid metabolism. However, it remains unclear how the L-cysteine-responsive Lrp/AsnC family regulator perceives and responds to L-cysteine. Here, we try to elucidate the molecular mechanism of the L-cysteine-responsive transcriptional regulator. Through 5'RACE and EMSA, we discovered a 15 bp incompletely complementary pair palindromic sequence essential for DecR binding, which differed slightly from the binding sequence of other Lrp/AsnC transcription regulators. Using alanine scanning, we identified the L-cysteine binding site on DecR and found that different Lrp/AsnC regulators adjust their binding pocket's side-chain residues to accommodate their specific effector. MD simulations were then conducted to explore how ligand binding influences the allosteric behavior of the protein. PCA and in silico docking revealed that ligand binding induced perturbations in the linker region, triggering conformational alterations and leading to the relocalization of the DNA-binding domains, enabling the embedding of the DNA-binding region of DecR into the DNA molecule, thereby enhancing DNA-binding affinity. Our findings can broaden the understanding of the recognition and regulatory mechanisms of the Lrp/AsnC-type transcription factors, providing a theoretical basis for further investigating the molecular mechanisms of other transcription factors.

The Intricacies of Renal Phosphate Reabsorption-An Overview.

To maintain an optimal body content of phosphorus throughout postnatal life, variable phosphate absorption from food must be finely matched with urinary excretion. This amazing feat is accomplished through synchronised phosphate transport by myriads of ciliated cells lining the renal proximal tubules. These respond in real time to changes in phosphate and composition of the renal filtrate and to hormonal instructions. How they do this has stimulated decades of research. New analytical techniques, coupled with incredible advances in computer technology, have opened new avenues for investigation at a sub-cellular level. There has been a surge of research into different aspects of the process. These have verified long-held beliefs and are also dramatically extending our vision of the intense, integrated, intracellular activity which mediates phosphate absorption. Already, some have indicated new approaches for pharmacological intervention to regulate phosphate in common conditions, including chronic renal failure and osteoporosis, as well as rare inherited biochemical disorders. It is a rapidly evolving field. The aim here is to provide an overview of our current knowledge, to show where it is leading, and where there are uncertainties. Hopefully, this will raise questions and stimulate new ideas for further research.

Convergent High O2 Affinity but Distinct ATP-Mediated Allosteric Regulation of Hemoglobins in Oviparous and Viviparous Eremias Lizards from the Qinghai-Tibet Plateau.

The functional adaptation and underlying molecular mechanisms of hemoglobins (Hbs) have primarily concentrated on mammals and birds, with few reports on reptiles. This study aimed to investigate the convergent and species-specific high-altitude adaptation mechanisms of Hbs in two Eremias lizards from the Qinghai-Tibet Plateau. The Hbs of high-altitude E. argus and E. multiocellata were characterized by significantly high overall and intrinsic Hb-O2 affinity compared to their low-altitude populations. Despite the similarly low Cl- sensitivities, the Hbs of high-altitude E. argus exhibited higher ATP sensitivity and ATP-dependent Bohr effects than that of E. multiocellata, which could facilitate O2 unloading in respiring tissues. Eremias lizards Hbs exhibited similarly low temperature sensitivities and relatively high Bohr effects at lower temperatures, which could help to stably deliver and release O2 to cold extremities at low temperatures. The oxygenation properties of Hbs in high-altitude populations might be attributed to varying ratios of ß2/ß1 globin and substitutions on the ß2-type globin. Notably, the Asn12Ala in lowland E. argus could cause localized destabilization of the E-helix in the tetrameric Hb by elimination of hydrogen bonds, thereby resulting in its lowest O2 affinity. This study provides a valuable reference for the high-altitude adaptation mechanisms of hemoglobins in reptiles.

Insights into the regulation of malate dehydrogenase: inhibitors, activators, and allosteric modulation by small molecules.

Cellular metabolism comprises a complex network of biochemical anabolic and catabolic processes that fuel the growth and survival of living organisms. The enzyme malate dehydrogenase (MDH) is most known for its role in oxidizing malate to oxaloacetate (OAA) in the last step of the tricarboxylic acid (TCA) cycle, but it also participates in the malate-aspartate shuttle in the mitochondria as well as the glyoxylate cycle in plants. These pathways and the specific reactions within them are dynamic and must be carefully calibrated to ensure a balance between nutrient/energy supply and demand. MDH structural and functional complexity requires a variety of regulatory mechanisms, including allosteric regulation, feedback, and competitive inhibition, which are often dependent on whether the enzyme is catalyzing its forward or reverse reaction. Given the role of MDH in central metabolism and its potential as a target for therapeutics in both cancer and infectious diseases, there is a need to better understand its regulation. The involvement of MDH in multiple pathways makes it challenging to identify which effectors are critical to its activity. Many of the in vitro experiments examining MDH regulation were done decades ago, and though allosteric sites have been proposed, none to date have been specifically mapped. This review aims to provide an overview of the current knowledge surrounding MDH regulation by its substrate, products, and other intermediates of the TCA cycle while highlighting all the gaps in our understanding of its regulatory mechanisms.

Dynamically driven correlations in elastic net models reveal sequence of events and causality in proteins.

An explicit analytic solution is given for the Langevin equation applied to the Gaussian Network Model of a protein subjected to both a random and a deterministic periodic force. Synchronous and asynchronous components of time correlation functions are derived and an expression for phase differences in the time correlations of residue pairs is obtained. The synchronous component enables the determination of dynamic communities within the protein structure. The asynchronous component reveals causality, where the time correlation function between residues i and j differs depending on whether i is observed before j or vice versa, resulting in directional information flow. Driver and driven residues in the allosteric process of cyclophilin A and human NAD-dependent isocitrate dehydrogenase are determined by a perturbation-scanning technique. Factors affecting phase differences between fluctuations of residues, such as network topology, connectivity, and residue centrality, are identified. Within the constraints of the isotropic Gaussian Network Model, our results show that asynchronicity increases with viscosity and distance between residues, decreases with increasing connectivity, and decreases with increasing levels of eigenvector centrality.

Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca2+ binding, altering transport Ca2+ dependence without actually affecting the equilibrium binding affinity of the Ca2+-coordinating sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca2+ affinity of SERCA by interrupting allosteric activation of the pump by ATP.

Phosphorylation-dependent allosteric regulation of Cx43 gap junction inhibitor potency.

Physiological and pathological processes such as homeostasis, embryogenesis, development, tumorigenesis, and cell movement depend on the intercellular communication through gap junctions (GJIC). Connexin (Cx)-based GJ channels are formed of two apposing hemichannels in the contiguous cells and provide a direct pathway for electrical and metabolic intercellular communication. The main modulators of GJ conductance are transjunctional voltage, intracellular pH, Ca2+, Mg2+, and phosphorylation. Chemical modulators of GJIC are being used in cases of various intercellular communication-dependent diseases. In this study, we used molecular docking, dual whole-cell patch-clamp, and Western blotting to investigate the impact of connexin phosphorylation on GJ chemical gating by α-pinene and other GJ inhibitors (octanol, carbenoxolone, mefloquine, intracellular pH, glycyrrhetinic acid, and sevoflurane) in HeLa cells expressing exogenous Cx43 (full length and truncated at amino acid 258) and other connexins typical of heart and/or nervous system (Cx36, Cx40, Cx45, and Cx47), and in cells expressing endogenous Cx43 (Novikoff and U-87). We found that Ca2+-regulated kinases, such as Ca2+/calmodulin-dependent kinase II, atypical protein kinase C, cyclin-dependent kinase, and Pyk2 kinase may allosterically modulate the potency of α-pinene through phosphorylation of Cx43 C-terminus. The identified new phenomenon was Cx isoform-, inhibitor-, and cell type-dependent. Overall, these results suggest that compounds, the potency of which depends on receptor phosphorylation, might be of particular interest in developing targeted therapies for diseases accompanied by high kinase activity, such as cardiac arrhythmias, epilepsy, stroke, essential tremor, inflammation, and cancer.

Development of Allosteric Ribozymes for ATP and l-Histidine Based on the R3C Ligase Ribozyme.

During the evolution of the RNA, short RNAs are thought to have joined together to form long RNAs, enhancing their function as ribozymes. Previously, the artificial R3C ligase ribozyme (73 nucleotides) was successfully reduced to 46 nucleotides; however, its activity decreased significantly. Therefore, we aimed to develop allosteric ribozymes, whose activities could be regulated by effector compounds, based on the reduced R3C ligase ribozyme (R3C-A). Among the variants prepared by fusing an ATP-binding aptamer RNA with R3C-A, one mutant showed increased ligation activity in an ATP-dependent manner. Melting temperature measurements of the two RNA mutants suggested that the region around the aptamer site was stabilized by the addition of ATP. This resulted in a suitable conformation for the reaction at the ligation site. Another ribozyme was prepared by fusing R3C-A with a l-histidine-binding aptamer RNA, and the ligase activity increased with increasing l-histidine concentrations. Both ATP and l-histidine play prominent roles in current molecular biology and the interaction of RNAs and these molecules could be a key step in the evolution of the world of RNAs. Our results suggest promise in the development of general allosteric ribozymes that are independent of the type of effector molecule and provide important clues to the evolution of the RNA world.

Evolutionary Changes in Primate Glutamate Dehydrogenases 1 and 2 Influence the Protein Regulation by Ligands, Targeting and Posttranslational Modifications.

There are two paralogs of glutamate dehydrogenase (GDH) in humans encoded by the GLUD1 and GLUD2 genes as a result of a recent retroposition during the evolution of primates. The two human GDHs possess significantly different regulation by allosteric ligands, which is not fully characterized at the structural level. Recent advances in identification of the GDH ligand binding sites provide a deeper perspective on the significance of the accumulated substitutions within the two GDH paralogs. In this review, we describe the evolution of GLUD1 and GLUD2 after the duplication event in primates using the accumulated sequencing and structural data. A new gibbon GLUD2 sequence questions the indispensability of ancestral R496S and G509A mutations for GLUD2 irresponsiveness to GTP, providing an alternative with potentially similar regulatory features. The data of both GLUD1 and GLUD2 evolution not only confirm substitutions enhancing GLUD2 mitochondrial targeting, but also reveal a conserved mutation in ape GLUD1 mitochondrial targeting sequence that likely reduces its transport to mitochondria. Moreover, the information of GDH interactors, posttranslational modification and subcellular localization are provided for better understanding of the GDH mutations. Medically significant point mutations causing deregulation of GDH are considered from the structural and regulatory point of view.

Reconstruction the feedback regulation of amino acid metabolism to develop a non-auxotrophic L-threonine producing Corynebacterium glutamicum.

L-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation. The GRAS (generally regarded as safe) industrial workhorse Corynebacterium glutamicum is an attractive chassis for L-threonine production. However, the present L-threonine production in C. glutamicum cannot meet the requirement of industrialization due to the relatively low production level of L-threonine and the accumulation of large amounts of by-products (such as L-lysine, L-isoleucine, and glycine). Herein, to enhance the L-threonine biosynthesis in C. glutamicum, releasing the aspartate kinase (LysC) and homoserine dehydrogenase (Hom) from feedback inhibition by L-lysine and L-threonine, respectively, and overexpressing four flux-control genes were performed. Next, to reduce the formation of by-products L-lysine and L-isoleucine without the cause of an auxotrophic phenotype, the feedback regulation of dihydrodipicolinate synthase (DapA) and threonine dehydratase (IlvA) was strengthened by replacing the native enzymes with heterologous analogues with more sensitive feedback inhibition by L-lysine and L-isoleucine, respectively. The resulting strain maintained the capability of synthesizing enough amounts of L-lysine and L-isoleucine for cell biomass formation but exhibited almost no extracellular accumulation of these two amino acids. To further enhance L-threonine production and reduce the by-product glycine, L-threonine exporter and homoserine kinase were overexpressed. Finally, the rationally engineered non-auxotrophic strain ZcglT9 produced 67.63 g/L (17.2% higher) L-threonine with a productivity of 1.20 g/L/h (108.0% higher) in fed-batch fermentation, along with significantly reduced by-product accumulation, representing the record for L-threonine production in C. glutamicum. In this study, we developed a strategy of reconstructing the feedback regulation of amino acid metabolism and successfully applied this strategy to de novo construct a non-auxotrophic L-threonine producing C. glutamicum. The main end by-products including L-lysine, L-isoleucine, and glycine were almost eliminated in fed-batch fermentation of the engineered C. glutamicum strain. This strategy can also be used for engineering producing strains for other amino acids and derivatives.