Conditions for effective allosteric feedforward and feedback in metabolic pathways

Whether an allosteric feedback or feedforward modifier actually has an effect on the steady-state properties of a metabolic pathway depends not only on the allosteric modifier effect itself, but also on the control properties of the affected allosteric enzyme in the pathway of which it is part. Different modification mechanisms are analysed: mixed inhibition, allosteric inhibition and activation of the reversible Monod-Wyman-Changeux and reversible Hill models. In conclusion, it is shown that, whereas a modifier effect on substrate and product binding (specific effects) can be an effective negative feedback mechanism, it is much less effective as a positive feedforward mechanism. The prediction is that catalytic effects that change the apparent limiting velocity would be more effective in feedforward activation.     

Role of packing defects in the evolution of allostery and induced fit in human UDP-glucose dehydrogenase

Allosteric feedback inhibition is the mechanism by which metabolic end products regulate their own biosynthesis by binding to an upstream enzyme. Despite its importance in controlling metabolism, there are relatively few allosteric mechanisms understood in detail. This is because allostery does not have an identifiable structural motif, making the discovery of new allosteric enzymes a difficult process. The lack of a conserved motif implies that the evolution of each allosteric mechanism is unique. Here we describe an atypical allosteric mechanism in human UDP-α-d-glucose 6-dehydrogenase (hUGDH) based on an easily acquired and identifiable structural attribute: packing defects in the protein core. In contrast to classic allostery, the active and allosteric sites in hUGDH are present as a single, bifunctional site. Using two new crystal structures, we show that binding of the feedback inhibitor, UDP-α-d-xylose, elicits a distinct induced-fit response; a buried loop translates ～4 ? along and rotates ～180° about the main chain axis, requiring surrounding side chains to repack. This allosteric transition is facilitated by packing defects, which negate the steric conformational restraints normally imposed by the protein core. Sedimentation velocity studies show that this repacking favors the formation of an inactive hexameric complex with unusual symmetry. We present evidence that hUGDH and the unrelated enzyme dCTP deaminase have converged to very similar atypical allosteric mechanisms using the same adaptive strategy, the selection for packing defects. Thus, the selection for packing defects is a robust mechanism for the evolution of allostery and induced fit.     

The molecular basis of thrombin allostery revealed by a 1.8 A structure of the "slow" form

Thrombin participates in its own positive and negative feedback loops, and its allosteric state helps determine the hemostatic balance. Here we present the 1.8 A crystallographic structure of S195A thrombin in two conformational states: active site occupied and active site free. The active site-occupied form shows how thrombin can accommodate substrates, such as protein C. The active site-free form is in a previously unobserved closed conformation of thrombin, which satisfies all the conditions of the so-called "slow" form. A mechanism of allostery is revealed, which relies on the concerted movement of the disulphide bond between Cys168 and 182 and aromatic residues Phe227, Trp215, and Trp60d. These residues constitute an allosteric switch, which is flipped directly through sodium binding, resulting in the fast form with an open active site.     

Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition

Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.     

Regulation of the aromatic pathway in the cyanobacterium Synechococcus sp. strain Pcc6301 (Anacystis nidulans).

A pattern of allosteric control for aromatic biosynthesis in cyanobacteria relies upon early-pathway regulation as the major control point for the entire branched pathway. In Synechococcus sp. strain PCC6301 (Anacystis nidulans), two enzymes which form precursors for L-phenylalanine biosynthesis are subject to control by feedback inhibition. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (first pathway enzyme) is feedback inhibited by L-tyrosine, whereas prephenate dehydratase (enzyme step 9) is feedback inhibited by L-phenylalanine and allosterically activated by L-tyrosine. Mutants lacking feedback inhibition of prephenate dehydratase excreted relatively modest quantities of L-phenylalanine. In contrast, mutants deregulated in allosteric control of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase excreted large quantities of L-phenylalanine (in addition to even greater quantities of L-tyrosine). Clearly, in the latter mutants, the elevated levels of prephenate must overwhelm the inhibition of prephenate dehydratase by L-phenylalanine, an effect assisted by increased intracellular L-tyrosine, an allosteric activator. The results show that early-pathway flow regulated in vivo by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is the dominating influence upon metabolite flow-through to L-phenylalanine. L-Tyrosine biosynthesis exemplifies such early-pathway control even more simply, since 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is the sole regulatory enzyme subject to end-product control by L-tyrosine.     

Glycogen synthase Hymenolepis diminuta. I. Allosteric activation and inhibition.

Glycogen synthase (UDP glucose: glycogen alpha-4-glycosyltransferase, EC2.4.1.11) of the tapeworm Hymenolepis diminuta exists in 2 forms: 1) the I-form (independent), which has significant activity in the absence of glucose 6-phosphate (G6P); and 2) the phosphorylated D-form (dependent), which has no enzymatic activity unless G6P is present. The activity of the I-form is greatly enhanced by a variety of allosteric effectors which have, as their common feature, 1 or more phosphate groups. These include inorganic phosphate (Pi), several sugar phosphates, some phosphorylated glycolytic intermediates, and nucleoside mono- and triphosphates. Competition studies suggest that while most of the positive effectors act at the same site on the enzyme (the "G6P site"), fructose 1,6-diphosphate (FDP) and 2,3-diphosphoglyceric acid (2,3DPG) act at low concentrations to stimulate the enzyme at another locus (the "diphosphate site"), while at high concentrations they competitively inhibit the binding of G6P and of the other activators. The inhibition by high uridine monophosphate (UMP) concentrations is competitive only with the activator uridine triphosphate (UTP), suggesting the existence of a third type of allosteric site (the "uridine nucleotide site"). This third site may be the locus for feedback inhibition by the product uridine diphosphate (UDP), a control mechanism which has been observed to occur in mammalian systems. The allosteric control of the D-form of the enzyme is comparatively simple, apparently involving only one site (the "G6P site") that binds a few effects with greatly reduced affinity. Pi reverses the activation of the D-form by G6P.     

Structure of N-acetyl-l-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor l-arginine bound

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in l-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by l-arginine, although l-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by l-arginine, we have determined the structure of the mmNAGS/K complexed with l-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of l-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the l-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when l-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by l-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism.     

The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter

The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of d- and l-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by l-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well.     

Taxonomic implications of temperature dependence of the allosteric inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase in Bacillus

The qualitative pattern of control for 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase is a stable and strongly conserved trait of related bacteria and constitutes a reliable generic character. In Bacillus, the generic control pattern for DAHP synthetase is sequential feedback inhibition, a regulatory pattern in which branch-point metabolites are feedback inhibitors. Member species of this genus have DAHP synthetases which vary quantitatively in the effect of temperature upon the sensitivity of the enzyme to feedback inhibition by prephenate. The magnitude of this temperature effect has been expressed quantitatively as the allosteric temperature ratio. The species clusters definable by allosteric temperature ratios correlate exceedingly well with subgroups previously distinguished on the basis of sporangial structure. Hence, two independently derived arrangements of Bacillus subgroups, depending upon very different methodologies, matched for all but 3 of the 24 species considered. The taxonomic position of these subgroups of the genus Bacillus is discussed.     

Differentially Regulated Isozymes of 3-Deoxy-d-arabino-Heptulosonate-7-Phosphate Synthase from Seedlings of Vigna radiata [L.] Wilczek

Two isozymes of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) designated DS-Mn and DS-Co were separated from seedlings of Vigna radiata [L.] Wilczek by DEAE-cellulose column chromatography. DS-Mn was activated 2.6-fold by 0.4 millimolar manganese, had an activity optimum of 7.0, and was substrate inhibited by erythrose-4-phosphate (E4P) concentrations in excess of 0.5 millimolar. In contrast, DS-Co had an activity optimum at pH 8.8, required E4P concentrations of at least 4 millimolar to approach saturation, and exhibited an absolute requirement for divalent cation (cobalt being the best). Regulatory properties of the two isozymes differed dramatically. The activity of DS-Mn was activated by chorismate (noncompetitively against E4P and competitively against phosphoenolpyruvate), and was inhibited by prephenate and l-arogenate (competitively against E4P and noncompetitively against phosphoenolpyruvate in both cases). Under standard assay conditions, l-arogenate inhibited the activity of DS-Mn 50% at a concentration of 155 micromolar and was at least 3 times more potent than prephenate on a molar basis. Weak inhibition of DS-Mn by l-tryptophan was also observed, the magnitude of inhibition increasing with decreasing pH. The pattern of allosteric control found for DS-Mn is consistent with the operation of a control mechanism of sequential feedback inhibition governing overall pathway flux. DS-Co was not subject to allosteric control by any of the molecules affecting DS-Mn. However, DS-Co was inhibited by caffeate (3,4-dihydroxycinnamate), noncompetitively with respect to either substrate. The striking parallels between the isozyme pairs of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase and chorismate mutase are noted—one isozyme in each case being tightly regulated, the other being essentially unregulated.     

Structures of R- and T-state Escherichia coli aspartokinase III. Mechanisms of the allosteric transition and inhibition by lysine

Aspartokinase III (AKIII) from Escherichia coli catalyzes an initial commitment step of the aspartate pathway, giving biosynthesis of certain amino acids including lysine. We report crystal structures of AKIII in the inactive T-state with bound feedback allosteric inhibitor lysine and in the R-state with aspartate and ADP. The structures reveal an unusual configuration for the regulatory ACT domains, in which ACT2 is inserted into ACT1 rather than the expected tandem repeat. Comparison of R- and T-state AKIII indicates that binding of lysine to the regulatory ACT1 domain in R-state AKIII instigates a series of changes that release a "latch", the beta15-alphaK loop, from the catalytic domain, which in turn undergoes large rotational rearrangements, promoting tetramer formation and completion of the transition to the T-state. Lysine-induced allosteric transition in AKIII involves both destabilizing the R-state and stabilizing the T-state tetramer. Rearrangement of the catalytic domain blocks the ATP-binding site, which is therefore the structural basis for allosteric inhibition of AKIII by lysine.     

Aspartokinase of Myxococcus xanthus: "feedback stimulation" by required amino acids

The aspartokinase activity found in extracts of the bacterium Myxococcus xanthus was subject to feedback inhibition and feedback repression by l-threonine and l-lysine. Both types of inhibition were essentially additive. The required amino acids, l-isoleucine and l-methionine, caused considerable increase in the activity of the enzyme. This phenomenon is referred to as "feedback stimulation." The polyamine, spermidine, exerted strong enhancement of the activity even at 0.1 mM. Meso-diaminopimelate, although not inhibitory by itself, abolished the activation exerted by either l-isoleucine or l-methionine. The possible physiological significance of interactions between the various effectors is discussed.     

Exploring a potential allosteric inhibition mechanism in the motor domain of human Eg-5

Kinesin-5 (Eg-5), microtubule motor protein, is one of the emerging drug targets in cancer research. Several inhibitors have been reported to bind the hEg-5 “motor domain” in two different locations that are potentially allosteric. Interestingly, the crystal structure of Eg-5 bound to benzimidazole unveils two chemically different allosteric pockets (PDB ID: 3ZCW). The allosteric modulators inhibit Eg-5 activity by causing conformational changes that affect nucleotide turnover rate. In the present work, three allosteric inhibitors were simulated along with the substrate nucleotides (ADP and ATP) to capture conformation changes induced by the allosteric inhibitors. To analyze the allosteric inhibition mechanism, we used dynamics cross-correlation, principal component analysis (PCA), and enthalpic calculations. The loop L5 interaction is determined by the type of substrate bind at the nucleotide binding site. The SW-II flexibility increased upon dual allosteric inhibition by SB-743921 and 6a. The ionic interaction between R221-E116 is observed only in the presence of two allosteric inhibitors. Also, we noticed that the α2/α3 helical orientation is responsible for the SW-1 loop position and substrate binding. Our simulation data suggest the critical chemical features required to block the motor domain by the allosteric inhibitors. The results summarized in this work will help the researchers to design better therapeutic agents targeting hEg-5.

Communicated by Ramaswamy H. Sarma     

Enzymological basis for growth inhibition by L-phenylalanine in the cyanobacterium Synechocystis sp. 29108

The pattern of allosteric control in the biosynthetic pathway for aromatic amino acids provides a basis to explain vulnerability to growth inhibition by l-phenylalanine (0.2 mM or greater) in the unicellular cyanobacterium Synechocystis sp. 29108. We attribute growth inhibition to the hypersensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase to feedback inhibition by l-phenylalanine. Hyperregulation of this initial enzyme of aromatic biosynthesis depletes the supply of precursors needed for biosynthesis of l-tyrosine and l-tryptophan. Consistent with this mechanism is the total reversal of phenylalanine inhibition by a combination of tyrosine and tryptophan. Inhibited cultures also contained decreased levels of phycocyanin pigments, a characteristic previously correlated with amino acid starvation in cyanobacteria. l-Phenylalanine is a potent noncompetitive inhibitor (with both substrates) of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase, whereas l-tyrosine is a very weak inhibitor. Prephenate dehydratase also displays allosteric sensitivity to phenylalanine (inhibition) and to tyrosine (activation). Both 2-fluoro and 4-fluoro derivatives of phenylalanine were potent analog antimetabolites, and these were used in addition to l-phenylalanine as selective agents for resistant mutants. Mutants were isolated which excreted both phenylalanine and tyrosine, the consequence of an altered 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase no longer sensitive to feedback inhibition. Simultaneous insensitivity to l-tyrosine suggests that l-tyrosine acts as a weak analog mimic of l-phenylalanine at a common binding site. Prephenate dehydratase in the regulatory mutants was unaltered. Surprisingly, in view of the lack of regulation in the tyrosine branchlet of the pathway, such mutants excrete more phenylalanine than tyrosine, indicating that l-tyrosine activation dominates l-phenylalanine inhibition of prephenate dehydratase in vivo. In mutant Phe r19 the loss in allosteric sensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase was accompanied by a threefold increase in specific activity. This could suggest that existence of a modest degree of repression control (autogenous) over 3-deoxy-d-arabinoheptulosonate synthase, although other explanations are possible. Specific activities of chorismate mutase, prephenate dehydratase, shikimate/nicotinamide adenine dinucleotide phosphate dehydrogenase, and arogenate/nicotinamide adenine dinucleotide phosphate dehydrogenase in mutant Phe r19 were identical with those of the wild type.     

Structural and functional characterization of α-isopropylmalate synthase and citramalate synthase, members of the LeuA dimer superfamily

The manipulation of modular regulatory domains from allosteric enzymes represents a possible mechanism to engineer allostery into non-allosteric systems. Currently, there is insufficient understanding of the structure/function relationships in modular regulatory domains to rationally implement this methodology. The LeuA dimer regulatory domain represents a well-conserved, novel fold responsible for the regulation of two enzymes involved in branched chain amino acid biosynthesis, α-isopropylmalate synthase and citramalate synthase. The LeuA dimer regulatory domain is responsible for the feedback inhibition of these enzymes by their respective downstream products. Both enzymes display multidomain architecture with a conserved N-terminal TIM barrel catalytic domain and a C-terminal (βββα)2 LeuA dimer domain joined by a flexible linker region. Due to the similarity of three-dimensional structure and catalytic mechanism combined with low sequence similarity, we propose these enzymes can be classified as members of the LeuA dimer superfamily. Despite their similarity, members of the LeuA dimer superfamily display diversity in their allosteric mechanisms. In this review, structural aspects of the LeuA dimer superfamily are discussed followed by three examples highlighting the diversity of allosteric mechanisms in the LeuA dimer superfamily.     

Kinetic studies of thymidine phosphorylase from mouse liver

Initial velocity and product inhibition studies of thymidine phosphorylase from mouse liver revealed that the basic reaction mechanism of this enzyme is a rapid equilibrium random bi-bi mechanism with an enzyme-phosphate-thymine dead-end complex. Thymine displayed both substrate inhibition and nonlinear product inhibition, i.e., slope and intercept replots vs. 1/[thymine] were nonlinear, indicating that there is more than one binding site on the enzyme for thymine and that when thymine is bound to one of these sites, the enzyme is inhibited. Furthermore, both thymidine and phosphate showed "cooperative effects" in the presence of thymine at concentrations above 60 microM, suggesting that the enzyme may have multiple interacting allosteric and/or catalytic sites. The deoxyribosyl transferase reaction catalyzed by this enzyme is phosphate-dependent, requires nonstoichiometric amounts of phosphate, and can proceed by an "enzyme-bound" 2-deoxyribose 1-phosphate intermediate. These findings are in accord with the rapid equilibrium random bi-bi mechanism and demonstrate that deoxyribosyl transfer by this enzyme involves an indirect-transfer mechanism. These results strongly suggest that phosphorolysis and deoxyribosyl transfer are catalyzed by the same site on thymidine phosphorylase.     

Integrating molecular dynamics and co-evolutionary analysis for reliable target prediction and deregulation of the allosteric inhibition of aspartokinase for amino acid production

Deregulation of allosteric inhibition of enzymes is a challenge for strain engineering and has been achieved so far primarily by random mutation and trial-and-error. In this work, we used aspartokinase, an important allosteric enzyme for industrial amino acids production, to demonstrate a predictive approach that combines protein dynamics and evolution for a rational reengineering of enzyme allostery. Molecular dynamic simulation of aspartokinase III (AK3) from Escherichia coli and statistical coupling analysis of protein sequences of the aspartokinase family allowed to identify a cluster of residues which are correlated during protein motion and coupled during the evolution. This cluster of residues forms an interconnected network mediating the allosteric regulation, including most of the previously reported positions mutated in feedback insensitive AK3 mutants. Beyond these mutation positions, we have successfully constructed another twelve targeted mutations of AK3 desensitized toward lysine inhibition. Six threonine-insensitive mutants of aspartokinase I-homoserine dehydrogenase I (AK1-HD1) were also created based on the predictions. The proposed approach can be widely applied for the deregulation of other allosteric enzymes.     

"Ping-pong" interactions between mitochondrial tRNA import receptors within a multiprotein complex

The mitochondrial genomes of a wide variety of species contain an insufficient number of functional tRNA genes, and translation of mitochondrial mRNAs is sustained by import of nucleus-encoded tRNAs. In Leishmania, transfer of tRNAs across the inner membrane can be regulated by positive and negative interactions between them. To define the factors involved in such interactions, a large multisubunit complex (molecular mass, approximately 640 kDa) from the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania, consisting of approximately 130-A particles, was isolated. The complex, when incorporated into phospholipid vesicles, induced specific, ATP- and proton motive force-dependent transfer of Leishmania tRNA(Tyr) as well as of oligoribonucleotides containing the import signal YGGYAGAGC. Moreover, allosteric interactions between tRNA(Tyr) and tRNA(Ile) were observed in the RNA import complex-reconstituted system, indicating the presence of primary and secondary tRNA binding sites within the complex. By a combination of antibody inhibition, photochemical cross-linking, and immunoprecipitation, it was shown that binding of tRNA(Ile) to a 21-kDa component of the complex is dependent upon tRNA(Tyr), while binding of tRNA(Tyr) to a 45-kDa component is inhibited by tRNA(Ile). This "ping-pong" mechanism may be an effective means to maintain a balanced tRNA pool for mitochondrial translation.     

Synergistic inhibition of Escherichia coli aspartate transcarbamylase by CTP and UTP: binding studies using continuous-flow dialysis.

The allosteric control of Escherichia coli aspartate transcarbamylase (ATCase) involves feedback inhibition by both CTP and UTP, although it is only in the presence of CTP that UTP appears to inhibit the activity of the enzyme. In order to better understand the parts played by both pyrimidine nucleotides in this synergistic inhibition, binding studies were performed by continuous-flow dialysis and ultracentrifugation methods. The results obtained show that UTP binds to ATCase in the absence of CTP. Nevertheless, this binding does not induce any inhibition unless CTP is present. The mutual influence of CTP and UTP on their respective binding constants suggests that they bind to the same regulatory sites. However, the results obtained cannot be satisfactorily explained by a simple competition between the nucleotides, and it is shown that reciprocal affinity enhancements play a fundamental role. CTP enhances the affinity of UTP for the regulatory sites 80-fold, and conversely, UTP enhances the affinity of CTP 5-fold. Interestingly, the isolated regulatory subunits bind the two pyrimidine nucleotides following the same pattern as the entire enzyme. These observations indicate that the synergistic inhibition mechanism relies entirely on interactions between the two adjacent allosteric sites which belong to the same regulatory dimer.     

Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacterium glutamicum for lysine production

Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter L-lysine within 30 h of fed-batch fermentation in a bioreactor.