Heterologous overexpression and characterization of homoserine dehydrogenase from Paracoccidioides brasiliensis.

The enzyme Homoserine dehydrogenase from Paracoccidioides brasiliensis (PbHSD), an interesting enzyme in the search for new antifungal drugs against paracoccidioidomycosis, was expressed by E. coli. Thirty milligrams of PbHSD with 94% of purity were obtained per liter of culture medium. The analysis by CD spectroscopy indicates a composition of 45.5 ± 7.3% of α-helices and 10.5 ± 7.0% ß-strands. Gel filtration chromatography indicates a homodimer as biological unity. Fluorescence emission spectroscopy has shown stability of PbHSD in the presence of urea until Cm of 4.13 ± 0.21 M, and a broad pH range in which there is no conformational change. The protein analysis by differential scanning calorimetry indicates high stability at room temperature, but low stability at high temperatures, suffering irreversible denaturation, with Tm = 58.65 ± 0.87 °C. Kinetic studies of PbHSD by molecular absorption spectroscopy in UV/Vis have shown an optimum pH between 9.35 and 9.50, with Michaelian behavior, presenting KM of 224 ± 15 µM and specific activity at optimum pH of 2.10 ± 0.07 µmol/min/mg for homoserine. Therefore, protein expression and purification were efficient, and the structural characterization has shown that PbHSD presents native conformation with enzymatic activity in kinetic assays.

Conformational changes in the catalytic region are responsible for heat-induced activation of hyperthermophilic homoserine dehydrogenase.

When overexpressed as an immature enzyme in the mesophilic bacterium Escherichia coli, recombinant homoserine dehydrogenase from the hyperthermophilic archaeon Sulfurisphaera tokodaii (StHSD) was markedly activated by heat treatment. Both the apo- and holo-forms of the immature enzyme were successively crystallized, and the two structures were determined. Comparison among the structures of the immature enzyme and previously reported structures of mature enzymes revealed that a conformational change in a flexible part (residues 160-190) of the enzyme, which encloses substrates within the substrate-binding pocket, is smaller in the immature enzyme. The immature enzyme, but not the mature enzyme, formed a complex that included NADP+, despite its absence during crystallization. This indicates that the opening to the substrate-binding pocket in the immature enzyme is not sufficient for substrate-binding, efficient catalytic turnover or release of NADP+. Thus, specific conformational changes within the catalytic region appear to be responsible for heat-induced activation.

Bayesian Inference for Integrating Yarrowia lipolytica Multiomics Datasets with Metabolic Modeling.

Optimizing the metabolism of microbial cell factories for yields and titers is a critical step for economically viable production of bioproducts and biofuels. In this process, tuning the expression of individual enzymes to obtain the desired pathway flux is a challenging step, in which data from separate multiomics techniques must be integrated with existing biological knowledge to determine where changes should be made. Following a design-build-test-learn strategy, building on recent advances in Bayesian metabolic control analysis, we identify key enzymes in the oleaginous yeast Yarrowia lipolytica that correlate with the production of itaconate by integrating a metabolic model with multiomics measurements. To this extent, we quantify the uncertainty for a variety of key parameters, known as flux control coefficients (FCCs), needed to improve the bioproduction of target metabolites and statistically obtain key correlations between the measured enzymes and boundary flux. Based on the top five significant FCCs and five correlated enzymes, our results show phosphoglycerate mutase, acetyl-CoA synthetase (ACSm), carbonic anhydrase (HCO3E), pyrophosphatase (PPAm), and homoserine dehydrogenase (HSDxi) enzymes in rate-limiting reactions that can lead to increased itaconic acid production.

Biochemical characterization and redesign of the coenzyme specificity of a novel monofunctional NAD+-dependent homoserine dehydrogenase from the human pathogen Neisseria gonorrhoeae.

Gonorrhoea, caused by Neisseria gonorrhoeae, is a major global public health concern. Homoserine dehydrogenase (HSD), a key enzyme in the aspartate pathway, is a promising metabolic target against pathogenic infections. In this study, a monofunctional HSD from N. gonorrhoeae (NgHSD) was overexpressed in Escherichia coli and purified to >95% homogeneity for biochemical characterization. Unlike the classic dimeric structure, the purified recombinant NgHSD exists as a tetramer in solution. We determined the enzymatic activity of recombinant NgHSD for l-homoserine oxidation, which revealed that this enzyme was NAD+ dependent, with an approximate 479-fold (kcat/Km) preference for NAD+ over NADP+, and that optimal activity for l-homoserine oxidation occurred at pH 10.5 and 40 °C. At 800 mM, neither NaCl nor KCl increased the activity of NgHSD, in contrast to the behavior of several reported NAD+-independent homologs. Moreover, threonine did not markedly inhibit the oxidation activity of NgHSD. To gain insight into the cofactor specificity, site-directed mutagenesis was used to alter coenzyme specificity. The double mutant L45R/S46R, showing the highest affinity for NADP+, caused a shift in coenzyme preference from NAD+ to NADP+ by a factor of ~974, with a catalytic efficiency comparable with naturally occurring NAD+-independent homologs. Collectively, our results should allow the exploration of drugs targeting NgHSD to treat gonococcal infections and contribute to the prediction of the coenzyme specificity of novel HSDs.

Molecular and Enzymatic Features of Homoserine Dehydrogenase from Bacillus subtilis.

Homoserine dehydrogenase (HSD) catalyzes the reversible conversion of L-aspartate-4- semialdehyde to L-homoserine in the aspartate pathway for the biosynthesis of lysine, methionine, threonine, and isoleucine. HSD has attracted great attention for medical and industrial purposes due to its recognized application in the development of pesticides and is being utilized in the large scale production of L-lysine. In this study, HSD from Bacillus subtilis (BsHSD) was overexpressed in Escherichia coli and purified to homogeneity for biochemical characterization. We examined the enzymatic activity of BsHSD for L-homoserine oxidation and found that BsHSD exclusively prefers NADP+ to NAD+ and that its activity was maximal at pH 9.0 and in the presence of 0.4 M NaCl. By kinetic analysis, Km values for L-homoserine and NADP+ were found to be 35.08 ± 2.91 mM and 0.39 ± 0.05 mM, respectively, and the Vmax values were 2.72 ± 0.06 µmol/min-1 mg-1 and 2.79 ± 0.11 µmol/min-1 mg-1, respectively. The apparent molecular mass determined with size-exclusion chromatography indicated that BsHSD forms a tetramer, in contrast to the previously reported dimeric HSDs from other organisms. This novel oligomeric assembly can be attributed to the additional C-terminal ACT domain of BsHSD. Thermal denaturation monitoring by circular dichroism spectroscopy was used to determine its melting temperature, which was 54.8°C. The molecular and biochemical features of BsHSD revealed in this study may lay the foundation for future studies on amino acid metabolism and its application for industrial and medical purposes.

New 4-methoxy-naphthalene derivatives as promisor antifungal agents for paracoccidioidomycosis treatment.

AIM: Novel 4-methoxy-naphthalene derivatives were synthesized based on hits structures in order to evaluate the antifungal activity against Paracoccidioides spp. METHODS: Antifungal activity of compounds was evaluated against P. brasiliensis and most promising compounds 2 and 3 were tested against eight clinically important fungal species. RESULTS: Compound 3 was the more active compound with MIC 8 to 32 µg.ml-1 for Paracoccidioides spp without toxicity monkey kidney and murine macrophagecells. Carbohydrazide 3 showed good synergistic antifungal activity with amphotericin B against P. brasiliensis specie. Titration assay of carbohydrazide 3 with PbHSD enzyme demonstrates the binding ligand-protein. Molecular dynamics simulations show that ligand 3 let the PbHSD protein more stable. CONCLUSION: New carbohydrazide 3 is an attractive lead for drug development to treat paracoccidioidomycoses.

The crystal structure of homoserine dehydrogenase complexed with l-homoserine and NADPH in a closed form.

Homoserine dehydrogenase from Thermus thermophilus (TtHSD) is a key enzyme in the aspartate pathway that catalyses the reversible conversion of l-aspartate-ß-semialdehyde to l-homoserine (l-Hse) with NAD(P)H. We determined the crystal structures of unliganded TtHSD, TtHSD complexed with l-Hse and NADPH, and Lys99Ala and Lys195Ala mutant TtHSDs, which have no enzymatic activity, complexed with l-Hse and NADP+ at 1.83, 2.00, 1.87 and 1.93 Å resolutions, respectively. Binding of l-Hse and NADPH induced the conformational changes of TtHSD from an open to a closed form: the mobile loop containing Glu180 approached to fix l-Hse and NADPH, and both Lys99 and Lys195 could make hydrogen bonds with the hydroxy group of l-Hse. The ternary complex of TtHSDs in the closed form mimicked a Michaelis complex better than the previously reported open form structures from other species. In the crystal structure of Lys99Ala TtHSD, the productive geometry of the ternary complex was almost preserved with one new water molecule taking over the hydrogen bonds associated with Lys99, while the positions of Lys195 and l-Hse were significantly retained with those of the wild-type enzyme. These results propose new possibilities that Lys99 is the acid-base catalytic residue of HSDs.

Inhibition of homoserine dehydrogenase by formation of a cysteine-NAD covalent complex.

Homoserine dehydrogenase (EC 1.1.1.3, HSD) is an important regulatory enzyme in the aspartate pathway, which mediates synthesis of methionine, threonine and isoleucine from aspartate. Here, HSD from the hyperthermophilic archaeon Sulfolobus tokodaii (StHSD) was found to be inhibited by cysteine, which acted as a competitive inhibitor of homoserine with a Ki of 11 µM and uncompetitive an inhibitor of NAD and NADP with Ki's of 0.55 and 1.2 mM, respectively. Initial velocity and product (NADH) inhibition analyses of homoserine oxidation indicated that StHSD first binds NAD and then homoserine through a sequentially ordered mechanism. This suggests that feedback inhibition of StHSD by cysteine occurs through the formation of an enzyme-NAD-cysteine complex. Structural analysis of StHSD complexed with cysteine and NAD revealed that cysteine situates within the homoserine binding site. The distance between the sulfur atom of cysteine and the C4 atom of the nicotinamide ring was approximately 1.9 Å, close enough to form a covalent bond. The UV absorption-difference spectrum of StHSD with and without cysteine in the presence of NAD, exhibited a peak at 325 nm, which also suggests formation of a covalent bond between cysteine and the nicotinamide ring.

Antifungal dipeptides incorporating an inhibitor of homoserine dehydrogenase.

The antifungal activity of 5-hydroxy-4-oxo-l-norvaline (HONV), exhibited under conditions mimicking human serum, may be improved upon incorporation of this amino acid into a dipeptide structure. Several HONV-containing dipeptides inhibited growth of human pathogenic yeasts of the Candida genus in the RPMI-1640 medium, with minimal inhibitory concentration values in the 32 to 64 µg mL-1 range. This activity was not affected by multidrug resistance that is caused by overexpression of genes encoding drug efflux proteins. The mechanism of antifungal action of HONV dipeptides involved uptake by the oligopeptide transport system, subsequent intracellular cleavage by cytosolic peptidases, and inhibition of homoserine dehydrogenase by the released HONV. The relative transport rates determined the anticandidal activity of HONV dipeptides.

Targeting the Homoserine Dehydrogenase of Paracoccidioides Species for Treatment of Systemic Fungal Infections.

This work evaluated new potential inhibitors of the enzyme homoserine dehydrogenase (HSD) of Paracoccidioides brasiliensis, one of the etiological agents of paracoccidioidomycosis. The tertiary structure of the protein bonded to the analogue NAD, and l-homoserine was modeled by homology. The model with the best output was subjected to gradient minimization, redocking, and molecular dynamics simulation. Virtual screening simulations with 187,841 molecules purchasable from the Zinc database were performed. After the screenings, 14 molecules were selected and analyzed by the use of absorption, distribution, metabolism, excretion, and toxicity criteria, resulting in four compounds for in vitro assays. The molecules HS1 and HS2 were promising, exhibiting MICs of 64 and 32 µg · ml-1, respectively, for the Pb18 isolate of P. brasilensis, 64 µg · ml-1 for two isolates of P. lutzii, and also synergy with itraconazole. The application of these molecules to human-pathogenic fungi confirmed that the HSD enzyme may be used as a target for the development of drugs with specific action against paracoccidioidomycosis; moreover, these compounds may serve as leads in the design of new antifungals.

Candida albicans Hom6 is a homoserine dehydrogenase involved in protein synthesis and cell adhesion.

BACKGROUND/PURPOSE: Candida albicans is a common fungal pathogen in humans. In healthy individuals, C. albicans represents a harmless commensal organism, but infections can be life threatening in immunocompromised patients. The complete genome sequence of C. albicans is extremely useful for identifying genes that may be potential drug targets and important for pathogenic virulence. However, there are still many uncharacterized genes in the Candida genome database. In this study, we investigated C. albicans Hom6, the functions of which remain undetermined experimentally. METHODS: HOM6-deleted and HOM6-reintegrated mutant strains were constructed. The mutant strains were compared with wild-type in their growth in various media and enzyme activity. Effects of HOM6 deletion on translation were further investigated by cell susceptibility to hygromycin B or cycloheximide, as well as by polysome profiling, and cell adhesion to polystyrene was also determined. RESULTS: C. albicans Hom6 exhibits homoserine dehydrogenase activity and is involved in the biosynthesis of methionine and threonine. HOM6 deletion caused translational arrest in cells grown under amino acid starvation conditions. Additionally, Hom6 protein was found in both cytosolic and cell-wall fractions of cultured cells. Furthermore, HOM6 deletion reduced C. albicans cell adhesion to polystyrene, which is a common plastic used in many medical devices. CONCLUSION: Given that there is no Hom6 homologue in mammalian cells, our results provided an important foundation for future development of new antifungal drugs.

Characterization of aspartate kinase and homoserine dehydrogenase from Corynebacterium glutamicum IWJ001 and systematic investigation of L-isoleucine biosynthesis.

Previously we have characterized a threonine dehydratase mutant TD(F383V) (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS(P176S, D426E, L575W) (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best L-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK(A279T) (encoded by lysC1) and a homoserine dehydrogenase mutant HD(G378S) (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK(A279T) is completely resistant to feed-back inhibition by L-threonine and L-lysine, and that HD(G378S) is partially resistant to L-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive L-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from L-lysine (decreased by 50.1 %) to L-threonine (4.85 g/L) with minor L-isoleucine and no L-homoserine accumulation, further co-expressing ilvA1 completely depleted L-threonine and strongly shifted carbon flux from L-lysine (decreased by 83.0 %) to L-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD(F383V) might be the main driving force for L-isoleucine over-synthesis in this case, and the partially feed-back resistant HD(G378S) might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.

Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases.

NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP, and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation. Structural analysis and site-directed mutagenesis showed that the large number of interactions between the cofactor and the enzyme are responsible for the lack of reactivity of the enzyme towards NADP. This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.

Structural basis for the catalytic mechanism of homoserine dehydrogenase.

Homoserine dehydrogenase (HSD) is an oxidoreductase in the aspartic acid pathway. This enzyme coordinates a critical branch point of the metabolic pathway that leads to the synthesis of bacterial cell-wall components such as L-lysine and m-DAP in addition to other amino acids such as L-threonine, L-methionine and L-isoleucine. Here, a structural rationale for the hydride-transfer step in the reaction mechanism of HSD is reported. The structure of Staphylococcus aureus HSD was determined at different pH conditions to understand the basis for the enhanced enzymatic activity at basic pH. An analysis of the crystal structure revealed that Lys105, which is located at the interface of the catalytic and cofactor-binding sites, could mediate the hydride-transfer step of the reaction mechanism. The role of Lys105 was subsequently confirmed by mutational analysis. Put together, these studies reveal the role of conserved water molecules and a lysine residue in hydride transfer between the substrate and the cofactor.

Metabolic and genetic factors affecting the productivity of pyrimidine nucleoside in Bacillus subtilis.

BACKGROUND: Cytidine and uridine are produced commercially by Bacillus subtilis. The production strains of cytidine and uridine were both derivatives from mutagenesis. However, the exact metabolic and genetic factors affecting the productivity remain unknown. Genetic engineering may be a promising approach to identify and confirm these factors. RESULTS: With the deletion of the cdd and hom genes, and the deregulation of the pyr operon in Bacillus subtilis168, the engineered strain produced 200.9 mg/L cytidine, 14.9 mg/L uridine and 960.1 mg/L uracil. Then, the overexpressed prs gene led to a dramatic increase of uridine by 25.9 times along with a modest increase of cytidine. Furthermore, the overexpressed pyrG gene improved the production of cytidine, uridine and uracil by 259.5%, 11.2% and 68.8%, respectively. Moreover, the overexpression of the pyrH gene increasesd the yield of cytidine by 40%, along with a modest augments of uridine and uracil. Lastly, the deletion of the nupC-pdp gene resulted in a doubled production of uridine up to 1684.6 mg/L, a 14.4% increase of cytidine to 1423 mg/L, and a 99% decrease of uracil to only 14.2 mg/L. CONCLUSIONS: The deregulation of the pyr operon and the overexpression of the prs, pyrG and pyrH genes all contribute to the accumulation of pyrimidine nucleoside compounds in the medium. Among these factors, the overexpression of the pyrG and pyrH genes can particularly facilitate the production of cytidine. Meanwhile, the deletion of the nupC-pdp gene can obviously reduce the production of uracil and simultaneously improve the production of uridine.

The contest for precursors: channelling L-isoleucine synthesis in Corynebacterium glutamicum without byproduct formation.

L-Isoleucine is an essential amino acid, which is required as a pharma product and feed additive. Its synthesis shares initial steps with that of L-lysine and L-threonine, and four enzymes of L-isoleucine synthesis have an enlarged substrate specificity involved also in L-valine and L-leucine synthesis. As a consequence, constructing a strain specifically overproducing L-isoleucine without byproduct formation is a challenge. Here, we analyze for consequences of plasmid-encoded genes in Corynebacterium glutamicum MH20-22B on L-isoleucine formation, but still obtain substantial accumulation of byproducts. In a different approach, we introduce point mutations into the genome of MH20-22B to remove the feedback control of homoserine dehydrogenase, hom, and threonine dehydratase, ilvA, and we assay sets of genomic promoter mutations to increase hom and ilvA expression as well as to reduce dapA expression, the latter gene encoding the dihydrodipicolinate synthase. The promoter mutations are mirrored in the resulting differential protein levels determined by a targeted LC-MS/MS approach for the three key enzymes. The best combination of genomic mutations was found in strain K2P55, where 53 mM L-isoleucine could be obtained. Whereas in fed-batch fermentations with the plasmid-based strain, 94 mM L-isoleucine with L-lysine as byproduct was formed; with the plasmid-less strain K2P55, 109 mM L-isoleucine accumulated with no substantial byproduct formation. The specific molar yield with the latter strain was 0.188 mol L-isoleucine (mol glucose)(-1) which characterizes it as one of the best L-isoleucine producers available and which does not contain plasmids.

Rational design of allosteric regulation of homoserine dehydrogenase by a nonnatural inhibitor L-lysine.

Allosteric proteins, which can sense different signals, are interesting biological parts for synthetic biology. In particular, the design of an artificial allosteric enzyme to sense an unnatural signal is both challenging and highly desired, for example, for a precise and dynamical control of fluxes of growth-essential but byproduct pathways in metabolic engineering of industrial microorganisms. In this work, we used homoserine dehydrogenase (HSDH) of Corynebacterium glutamicum, which is naturally allosterically regulated by threonine and isoleucine, as an example to demonstrate the feasibility of reengineering an allosteric enzyme to respond to an unnatural inhibitor L-lysine. For this purpose, the natural threonine binding sites of HSD were first predicted and verified by mutagenesis experiments. The threonine binding sites were then engineered to a lysine binding pocket. The reengineered HSD only responds to lysine inhibition but not to threonine. This is a significant step toward the construction of artificial molecular circuits for dynamic control of growth-essential byproduct formation pathway for lysine biosynthesis.

Exploring the molecular basis for selective binding of homoserine dehydrogenase from Mycobacterium leprae TN toward inhibitors: a virtual screening study.

Homoserine dehydrogenase (HSD) from Mycobacterium leprae TN is an antifungal target for antifungal properties including efficacy against the human pathogen. The 3D structure of HSD has been firmly established by homology modeling methods. Using the template, homoserine dehydrogenase from Thiobacillus denitrificans (PDB Id 3MTJ), a sequence identity of 40% was found and molecular dynamics simulation was used to optimize a reliable structure. The substrate and co-factor-binding regions in HSD were identified. In order to determine the important residues of the substrate (L-aspartate semialdehyde (L-ASA)) binding, the ASA was docked to the protein; Thr163, Asp198, and Glu192 may be important because they form a hydrogen bond with HSD through AutoDock 4.2 software. neuraminidaseAfter use of a virtual screening technique of HSD, the four top-scoring docking hits all seemed to cation-π ion pair with the key recognition residue Lys107, and Lys207. These ligands therefore seemed to be new chemotypes for HSD. Our results may be helpful for further experimental investigations.

[Heterologous expression and characterization of L200F/D215K mutant of homoserine dehydrogenase from Corynebacterium pekinense AS1.299].

OBJECTIVE: To obtain a new homoserine dehydrogenase with better properties from Corynebacterium pekinense by the spatial structure transfromation. METHODS: Double mutants L200F/D215A, L200F/D215E, L200F/D215G and L200F/D215K were constructed by site-directed mutagenesis and expressed in E. coli BL21. L200F/D215K was characterized for its highest catalytic efficiency and compared with that of L200F. RESULTS: The Vmax of L200F/D215K was 36.92 U/mg, 1.24 times as that of L200F. The optimum reaction temperature of L200F/D215K was 37 degrees C, 2 degrees C higher than that of L200F. The optimum pH of L200F/D215K was 7.5, the same as that of L200F. The half-life time of L200F/D215K under optimum temperature was 4.16 h and was 1.12 times as that of L200F. Both L200F/D215K and L200F had good resistance to organic solvents and metal ions. CONCLUSION: Through the spatial structure transformation, the enzymatic activity was increased, and the enzymology properties was optimized.

Regulatory mechanisms after short- and long-term perturbed lysine biosynthesis in the aspartate pathway: the need for isogenes in Arabidopsis thaliana.

The aspartate-derived amino acid pathway in plants is an intensively studied metabolic pathway, because of the biosynthesis of the four essential amino acids lysine, threonine, isoleucine and methionine. The pathway is mainly controlled by the key regulatory enzymes aspartate kinase (AK; EC 2.7.2.4), homoserine dehydrogenase (HSDH; EC 1.1.1.3) and 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7), formerly referred to as dihydrodipicolinate synthase (DHDPS). They are encoded by isoenzyme families and it is not known why such families are evolutionarily maintained. To gain more insight into the specific roles and regulation of the isoenzymes, we inhibited DHDPS in Arabidopsis thaliana with the chemical compound (N,N-dimethylglycinatoboranyloxycarbonylmethyl)-dimethylamine-borane (DDAB) and compared the short-term effects on the biochemical and biomolecular level to the long-term adaptations in dhdps knockout mutants. We found that DHDPS2 plays a crucial role in controlling lysine biosynthesis, thereby stabilizing flux through the whole aspartate pathway. Moreover, DHDPS2 was also shown to influence the threonine level to a large extent. In addition, the lysine-sensitive AKs, AKLYS1 and AKLYS3 control the short- and long-term responses to perturbed lysine biosynthesis in Arabidopsis thaliana.