Production and characterization of bifunctional enzymes. Domain swapping to produce new bifunctional enzymes in the aspartate pathway

The bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli catalyzes non-consecutive reactions in the aspartate pathway of amino acid biosynthesis. Both catalytic activities are subject to allosteric regulation by the end product amino acid L-threonine. To examine the kinetics and regulation of the enzymes in this pathway, each of these catalytic domains were separately expressed and purified. The separated catalytic domains remain active, with each of their catalytic activities enhanced in comparison to the native enzyme. The allosteric regulation of the kinase activity is lost, and regulation of the dehydrogenase activity is dramatically decreased in these separate domains. To create a new bifunctional enzyme that can catalyze consecutive metabolic reactions, the aspartokinase I domain was fused to the enzyme that catalyzes the intervening reaction in the pathway, aspartate semialdehyde dehydrogenase. A hybrid bifunctional enzyme was also created between the native monofunctional aspartokinase III, an allosteric enzyme regulated by lysine, and the catalytic domain of homoserine dehydrogenase I with its regulatory interface domain still attached. In this hybrid the kinase activity remains sensitive to lysine, while the dehydrogenase activity is now regulated by both threonine and lysine. The dehydrogenase domain is less thermally stable than the kinase domain and becomes further destabilized upon removal of the regulatory domain. The more stable aspartokinase III is further stabilized against thermal denaturation in the hybrid bifunctional enzyme and was found to retain some catalytic activity even at temperatures approaching 100 degrees C.     

An enzyme mechanism language for the mathematical modeling of metabolic pathways

MOTIVATION: As a first step toward the elucidation of the systems biology of complex biological systems, it was our goal to mathematically model common enzyme catalytic and regulatory mechanisms that repeatedly appear in biological processes such as signal transduction and metabolic pathways. RESULTS: We describe kMech, a Cellerator language extension that describes a suite of enzyme mechanisms. Each enzyme mechanism is parsed by kMech into a set of fundamental association-dissociation reactions that are translated by Cellerator into ordinary differential equations that are numerically solved by Mathematica. In addition, we present methods that use commonly available kinetic measurements to estimate rate constants required to solve these differential equations.     

A mathematical model for the branched chain amino acid biosynthetic pathways of Escherichia coli K12

As a first step toward the elucidation of the systems biology of the model organism Escherichia coli, it was our goal to mathematically model a metabolic system of intermediate complexity, namely the well studied end product-regulated pathways for the biosynthesis of the branched chain amino acids L-isoleucine, L-valine, and L-leucine. This has been accomplished with the use of kMech (Yang, C.-R., Shapiro, B. E., Mjolsness, E. D., and Hatfield, G. W. (2005) Bioinformatics 21, in press), a Cellerator (Shapiro, B. E., Levchenko, A., Meyerowitz, E. M., Wold, B. J., and Mjolsness, E. D. (2003) Bioinformatics 19, 677-678) language extension that describes a suite of enzyme reaction mechanisms. Each enzyme mechanism is parsed by kMech into a set of fundamental association-dissociation reactions that are translated by Cellerator into ordinary differential equations. These ordinary differential equations are numerically solved by Mathematica. Any metabolic pathway can be simulated by stringing together appropriate kMech models and providing the physical and kinetic parameters for each enzyme in the pathway. Writing differential equations is not required. The mathematical model of branched chain amino acid biosynthesis in E. coli K12 presented here incorporates all of the forward and reverse enzyme reactions and regulatory circuits of the branched chain amino acid biosynthetic pathways, including single and multiple substrate (Ping Pong and Bi Bi) enzyme kinetic reactions, feedback inhibition (allosteric, competitive, and non-competitive) mechanisms, the channeling of metabolic flow through isozymes, the channeling of metabolic flow via transamination reactions, and active transport mechanisms. This model simulates the results of experimental measurements.     

Identification of six novel allosteric effectors of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase isoforms. Physiological context sets the specificity

The Arabidopsis genome contains two genes predicted to code for bifunctional aspartate kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. We first characterized the kinetic and regulatory properties of the recombinant enzymes, showing that they mainly differ with respect to the inhibition of the homoserine dehydrogenase activity by threonine. A systematic search for other allosteric effectors allowed us to identify an additional inhibitor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, and valine) equally efficient on aspartate kinase I activity (4-fold activation). The six effectors of aspartate kinase I were all activators of aspartate kinase II activity (13-fold activation) and displayed a similar specificity for the enzyme. No synergy between different effectors could be observed. The activation, which resulted from a decrease in the Km values for the substrates, was detected using low substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the presence of physiological concentrations of the seven allosteric effectors confirmed that aspartate kinase I and II activities were highly sensitive to changes in alanine and threonine concentrations. Thus, physiological context rather than enzyme structure sets the specificity of the allosteric control. Stimulation by alanine may play the role of a feed forward activation of the aspartate-derived amino acid pathway in plant.     

Regulation of the fructose 6-phosphate/fructose 2,6-bisphosphate cycle by enzyme phosphorylation and sn-glycerol 3-phosphate

The regulation of the Fru-6-P/Fru-2,6-P2 cycle by the cooperation of allosteric and covalent mechanisms was investigated in a reconstituted enzyme system under in vitro conditions. Phosphorylation of the bifunctional enzyme exerts a much stronger effect than sn-glycerol 3-phosphate in lowering the quasi-stationary concentration of fructose 2,6-bisphosphate and in increasing the critical concentration of the fructose phosphates, respectively. However, sn-glycerol 3-phosphate is able to strongly amplify the decrease of the quasi-stationary concentration of fructose 2,6-bisphosphate due to phosphorylation. The experiments can be described by a mathematical model involving rate equations for the dephosphorylated and the phosphorylated PFD-2 and FBPase-2. The results are compared with data from the literature obtained under in vivo conditions.     

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.     

A second allosteric site in Escherichia coli aspartate transcarbamoylase

Escherichia coli aspartate transcarbamoylase is feedback inhibited by CTP and UTP in the presence of CTP. Here, we show by X-ray crystallography that UTP binds to a unique site on each regulatory chain of the enzyme that is near but not overlapping with the known CTP site. These results bring into question all of the previously proposed mechanisms of allosteric regulation in aspartate transcarbamoylase.     

Stabilization of the R allosteric structure of Escherichia coli aspartate transcarbamoylase by disulfide bond formation

Here we report the first use of disulfide bond formation to stabilize the R allosteric structure of Escherichia coli aspartate transcarbamoylase. In the R allosteric state, residues in the 240s loop from two catalytic chains of different subunits are close together, whereas in the T allosteric state they are far apart. By substitution of Ala-241 in the 240s loop of the catalytic chain with cysteine, a disulfide bond was formed between two catalytic chains of different subunits. The cross-linked enzyme did not exhibit cooperativity for aspartate. The maximal velocity was increased, and the concentration of aspartate required to obtain one-half the maximal velocity, [Asp](0.5), was reduced substantially. Furthermore, the allosteric effectors ATP and CTP did not alter the activity of the cross-linked enzyme. When the disulfide bonds were reduced by the addition of 1,4-dithio-dl-threitol the resulting enzyme had kinetic parameters very similar to those observed for the wild-type enzyme and regained the ability to be activated by ATP and inhibited by CTP. Small-angle x-ray scattering was used to verify that the cross-linked enzyme was structurally locked in the R state and that this enzyme after reduction with 1,4-dithio-dl-threitol could undergo an allosteric transition similar to that of the wild-type enzyme. The complete abolition of homotropic and heterotropic regulation from stabilizing the 240s loop in its closed position in the R state, which forms the catalytically competent active site, demonstrates the significance that the quaternary structural change and closure of the 240s loop has in the functional mechanism of aspartate transcarbamoylase.     

Understanding the regulation of aspartate metabolism using a model based on measured kinetic parameters

The aspartate‐derived amino‐acid pathway from plants is well suited for analysing the function of the allosteric network of interactions in branched pathways. For this purpose, a detailed kinetic model of the system in the plant model Arabidopsis was constructed on the basis of in vitro kinetic measurements. The data, assembled into a mathematical model, reproduce in vivo measurements and also provide non‐intuitive predictions. A crucial result is the identification of allosteric interactions whose function is not to couple demand and supply but to maintain a high independence between fluxes in competing pathways. In addition, the model shows that enzyme isoforms are not functionally redundant, because they contribute unequally to the flux and its regulation. Another result is the identification of the threonine concentration as the most sensitive variable in the system, suggesting a regulatory role for threonine at a higher level of integration.     

Allosteric control model of bone remodelling containing periodical modes

To help to understand the modelling process that occurs when a scaffold is implanted it is vital to understand the rather complex bone remodelling process prevalent in native bone. We have formulated a mathematical model that predicts osteoactivity both in scaffolds, as well as in bone in vivo and could set a basis for the more detailed allosteric models. The model is extended towards a bio-cybernetic vision of basic multicellular unit (BMU) action, when some of the regulation loops have been modified to reflect the allosteric control mechanisms, developed by Michaels-Menten, Hill, Koshland-Nemethy-Filmer, Monod-Wyman-Changeux. By implementation of this approach a four-dimensional system was obtained that shows steady cyclic behaviour using a wide range of constants with clear biological meaning. We have observed that a local steady state appears as a limiting cycle in multi-dimensional phase space and this is discussed in this paper. Physiological interpretation of this limiting four-dimension cycle possibly related to a conservative-like value has been proposed. Analysis and simulation of the model has shown an analogy between this conservative value, as a kind of substrate-energy regenerative potential of the bone remodelling system with a molecular nature, and to the classical physical value--energy. This dynamic recovery potential is directed against both mechanical and biomechanical damage to the bone. Furthermore, the current model has credibility when compared to the normal bone remodelling process. In the framework of widely recognised Hill mechanisms of allosteric regulation the cyclic attractor, described formerly for a pure cellular model, prevails for different forms of feedback control. This result indicates the viability of the proposed existence of a conservative value (analogous to energy) that characterises the recovery potential of the bone remodelling cycle. Linear stability analysis has been performed in order to determine the robustness of the basic state, however, additional work is required to study a wider range of constants.     

The structural basis for allosteric inhibition of a threonine-sensitive aspartokinase

The commitment step to the aspartate pathway of amino acid biosynthesis is the phosphorylation of aspartic acid catalyzed by aspartokinase (AK). Most microorganisms and plants have multiple forms of this enzyme, and many of these isofunctional enzymes are subject to feedback regulation by the end products of the pathway. However, the archeal species Methanococcus jannaschii has only a single, monofunctional form of AK. The substrate l-aspartate binds to this recombinant enzyme in two different orientations, providing the first structural evidence supporting the relaxed regiospecificity previously observed with several alternative substrates of Escherichia coli AK ( Angeles, T. S., Hunsley, J. R., and Viola, R. E. (1992) Biochemistry 31, 799-805 ). Binding of the nucleotide substrate triggers significant domain movements that result in a more compact quaternary structure. In contrast, the highly cooperative binding of the allosteric regulator l-threonine to multiple sites on this dimer of dimers leads to an open enzyme structure. A comparison of these structures supports a mechanism for allosteric regulation in which the domain movements induced by threonine binding causes displacement of the substrates from the enzyme, resulting in a relaxed, inactive conformation.     

Conversion of the noncooperative Bacillus subtilis aspartate transcarbamoylase into a cooperative enzyme by a single amino acid substitution.

Allosteric enzymes are part of a unique class of enzymes which regulate metabolic pathways. On the molecular level, allosteric regulation is the result of interactions between discrete binding sites on the enzyme. In order to accommodate these multiple binding sites, allosteric enzymes have evolved with oligomeric quaternary structures. However, only a few oligomeric enzymes are known to have regulatory interactions between binding sites. Is regulatory activity an inherent property of oligomeric enzymes? The trimeric Bacillus subtilis aspartate transcarbamoylase catalyzes the first committed step of the pyrimidine biosynthetic pathway and is not known to be a regulatory enzyme. When an alanine residue is substituted for the active-site residue Arg-99 by site-specific mutagenesis, the regulatory activity of homotropic substrate cooperativity (Hill coefficient of 1.5) is observed in the resulting mutant enzyme. These results suggest that homotropic regulation may have evolved by a relatively small number of mutations to an oligomeric enzyme.     

Stabilization of the relaxed state of aspartate transcarbamoylase by modification with a bifunctional reagent.

Native aspartate transcarbamoylase from Escherichia coli was modified with the bifunctional reagent tartaryl diazide in the presence of the substrate carbamoyl phosphate and the substrate analog succinate. The product had the same sedimentation coefficient as the native enzyme but showed a marked increase in affinity for the substrate aspartate with a hyperbolic saturation curve. The Michaelis constant for aspartate (7.4 mM) is similar to that estimated for the relaxed state of the enzyme. The high substrate affinity was not produced if modification was conducted in the absence of substrate analogs or with a monofunctional reagent. The modified enzyme was also desensitized towards the allosteric effectors ATP and CTP. It appears to represent a stabilized relaxed state whose conversion to the taut state is presumably prevented by cross-linking.     

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.     

Discovery of intramolecular signal transduction network based on a new protein dynamics model of energy dissipation

A novel approach to reveal intramolecular signal transduction network is proposed in this work. To this end, a new algorithm of network construction is developed, which is based on a new protein dynamics model of energy dissipation. A key feature of this approach is that direction information is specified after inferring protein residue-residue interaction network involved in the process of signal transduction. This enables fundamental analysis of the regulation hierarchy and identification of regulation hubs of the signaling network. A well-studied allosteric enzyme, E. coli aspartokinase III, is used as a model system to demonstrate the new method. Comparison with experimental results shows that the new approach is able to predict all the sites that have been experimentally proved to desensitize allosteric regulation of the enzyme. In addition, the signal transduction network shows a clear preference for specific structural regions, secondary structural types and residue conservation. Occurrence of super-hubs in the network indicates that allosteric regulation tends to gather residues with high connection ability to collectively facilitate the signaling process. Furthermore, a new parameter of propagation coefficient is defined to determine the propagation capability of residues within a signal transduction network. In conclusion, the new approach is useful for fundamental understanding of the process of intramolecular signal transduction and thus has significant impact on rational design of novel allosteric proteins.     

Allostery and cooperativity in Escherichia coli aspartate transcarbamoylase

The allosteric enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli has been the subject of investigations for approximately 50 years. This enzyme controls the rate of pyrimidine nucleotide biosynthesis by feedback inhibition, and helps to balance the pyrimidine and purine pools by competitive allosteric activation by ATP. The catalytic and regulatory components of the dodecameric enzyme can be separated and studied independently. Many of the properties of the enzyme follow the Monod, Wyman Changeux model of allosteric control thus E. coli ATCase has become the textbook example. This review will highlight kinetic, biophysical, and structural studies which have provided a molecular level understanding of how the allosteric nature of this enzyme regulates pyrimidine nucleotide biosynthesis.     

Detailed kinetics and regulation of mammalian NAD-linked isocitrate dehydrogenase

A mathematical model is presented to describe the catalytic mechanism of mammalian NAD-linked isocitrate dehydrogenase (NAD-IDH), a highly regulated enzyme in the tricarboxylic acid cycle, a crucial pathway in energy metabolism and biosynthesis. The mechanism accounts for allosteric regulation by magnesium-bound isocitrate and EGTA and calcium-bound ATP and ADP. The developed model is used to analyze kinetic data for the cardiac enzyme and to estimate kinetic parameter values. Since the kinetic mechanism is expressed in terms of chemical species (rather than biochemical reactants), the model explicitly accounts for the effects of biochemical state (ionic strength, pH, temperature, and metal cation concentration) on the kinetics. Because the substrate isocitrate competes with allosteric activators (ATP and ADP) and an inhibitor (EGTA) for metal ion cofactors (Ca(2+) and Mg(2+)), the observed kinetic relationships between reactants, activator and inhibitor concentrations, and catalytic flux are complex. Our analysis reveals that under physiological conditions, the ADP/ATP ratio plays a more significant role than Ca(2+) concentration in regulating the enzyme's activity. In addition, the enzyme is highly sensitive to Mg(2+) concentration in the physiological range, pointing to a potential regulatory role of [Mg(2+)] in mitochondrial energy metabolism.     

Reciprocal allostery arising from a bienzyme assembly controls aromatic amino acid biosynthesis in Prevotella nigrescens

Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bienzyme assembly comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactions of aromatic amino acid biosynthesis in the bacterium Prevotella nigrescens (PniDAH7PS), respectively. The interactions between these two distinct catalytic domains support functional interreliance within this bifunctional enzyme. The binding of prephenate, the product of CM-catalyzed reaction, to the CM domain is associated with a striking rearrangement of overall protein conformation that alters the interdomain interactions and allosterically inhibits the DAH7PS activity. Here, we have further investigated the complex allosteric communication demonstrated by this bifunctional enzyme. We observed allosteric activation of CM activity in the presence of all DAH7PS substrates. Using small-angle X-ray scattering (SAXS) experiments, we show that changes in overall protein conformations and dynamics are associated with the presence of different DAH7PS substrates and the allosteric inhibitor prephenate. Furthermore, we have identified an extended interhelix loop located in CM domain, loop_C320-F333, as a crucial segment for the interdomain structural and catalytic communications. Our results suggest that the dual-function enzyme PniDAH7PS contains a reciprocal allosteric system between the two enzymatic moieties as a result of this bidirectional interdomain communication. This arrangement allows for a complex feedback and feedforward system for control of pathway flux by connecting the initiation and branch point of aromatic amino acid biosynthesis.