RÉSUMÉ
Most QM-cluster models of enzymes are constructed based on X-ray crystal structures, which limits comparison to in vivo structure and mechanism. The active site of chorismate mutase from Bacillus subtilis and the enzymatic transformation of chorismate to prephenate is used as a case study to guide construction of QM-cluster models built first from the X-ray crystal structure, then from molecular dynamics (MD) simulation snapshots. The Residue Interaction Network ResidUe Selector (RINRUS) software toolkit, developed by our group to simplify and automate the construction of QM-cluster models, is expanded to handle MD to QM-cluster model workflows. Several options, some employing novel topological clustering from residue interaction network (RIN) information, are evaluated for generating conformational clustering from MD simulation. RINRUS then generates a statistical thermodynamic framework for QM-cluster modeling of the chorismate mutase mechanism via refining 250 MD frames with density functional theory (DFT). The 250 QM-cluster models sampled provide a mean ΔG of 10.3 ± 2.6 kcal mol-1 compared to the experimental value of 15.4 kcal mol-1 at 25 °C. While the difference between theory and experiment is consequential, the level of theory used is modest and therefore "chemical" accuracy is unexpected. More important are the comparisons made between QM-cluster models designed from the X-ray crystal structure versus those from MD frames. The large variations in kinetic and thermodynamic properties arise from geometric changes in the ensemble of QM-cluster models, rather from the composition of the QM-cluster models or from the active site-solvent interface. The findings open the way for further quantitative and reproducible calibration in the field of computational enzymology using the model construction framework afforded with the RINRUS software toolkit.
Sujet(s)
Bacillus subtilis , Chorismate mutase , Simulation de dynamique moléculaire , Thermodynamique , Chorismate mutase/composition chimique , Chorismate mutase/métabolisme , Bacillus subtilis/enzymologie , Cristallographie aux rayons X , Domaine catalytique , Théorie de la fonctionnelle de la densité , Théorie quantique , Acide chorismique/métabolisme , Acide chorismique/composition chimique , LogicielRÉSUMÉ
Chorismate mutase (CM) enzymes have long served as model systems for benchmarking new methods and tools in computational chemistry. Despite the enzymes' prominence in the literature, the extent of the roles that activation enthalpy and entropy play in catalyzing the conversion of chorismate to prephenate is still subject to debate. Knowledge of these parameters is a key piece in fully understanding the mechanism of chorismate mutases. Within this study, we utilize EVB/MD free energy perturbation calculations at a range of temperatures, allowing us to extract activation enthalpies and entropies from an Arrhenius plot of activation free energies of the reaction catalyzed by a monofunctional Bacillus subtilis CM and the promiscuous enzyme isochorismate pyruvate lyase of Pseudomonas aeruginosa. In comparison to the uncatalyzed reaction, our results show that both enzyme-catalyzed reactions exhibit a substantial reduction in activation enthalpy, while the effect on activation entropy is relatively minor, demonstrating that enzyme-catalyzed CM reactions are enthalpically driven. Furthermore, we observe that the monofunctional CM from B. subtilis more efficiently catalyzes this reaction than its promiscuous counterpart. This is supported by a structural analysis of the reaction pathway at the transition state, from which we identified key residues explaining the enthalpically driven nature of the reactions and also the difference in efficiencies between the two enzymes.
Sujet(s)
Chorismate mutase , Chorismate mutase/composition chimique , Chorismate mutase/métabolisme , Thermodynamique , Entropie , TempératureRÉSUMÉ
Unlike typical chorismate mutases, the enzyme from Mycobacterium tuberculosis (MtCM) has only low activity on its own. Remarkably, its catalytic efficiency kcat/Km can be boosted more than 100-fold by complex formation with a partner enzyme. Recently, an autonomously fully active MtCM variant was generated using directed evolution, and its structure was solved by X-ray crystallography. However, key residues were involved in crystal contacts, challenging the functional interpretation of the structural changes. Here, we address these challenges by microsecond molecular dynamics simulations, followed up by additional kinetic and structural analyses of selected sets of specifically engineered enzyme variants. A comparison of wild-type MtCM with naturally and artificially activated MtCMs revealed the overall dynamic profiles of these enzymes as well as key interactions between the C-terminus and the active site loop. In the artificially evolved variant of this model enzyme, this loop is preorganized and stabilized by Pro52 and Asp55, two highly conserved residues in typical, highly active chorismate mutases. Asp55 stretches across the active site and helps to appropriately position active site residues Arg18 and Arg46 for catalysis. The role of Asp55 can be taken over by another acidic residue, if introduced at position 88 close to the C-terminus of MtCM, as suggested by molecular dynamics simulations and confirmed by kinetic investigations of engineered variants.
Sujet(s)
Chorismate mutase , Mycobacterium tuberculosis , Chorismate mutase/composition chimique , Simulation de dynamique moléculaire , Plan de recherche , Cristallographie aux rayons XRÉSUMÉ
Proteins fluctuate between different conformations in solution, and these conformational fluctuations can be important for protein function and allosteric regulation. The chorismate mutase from Saccharomyces cerevisiae (ScCM), a key enzyme in the biosynthesis of aromatic amino acids, is allosterically activated and inhibited by tryptophan and tyrosine, respectively. It was initially proposed that in the absence of effector, ScCM fluctuates between activated R and inhibited T conformations according to the Monod-Wyman-Changeux (MWC) model, although a more complex regulation pattern was later suggested by mutagenesis and kinetic data. Here we used NMR relaxation dispersion experiments to understand the conformational fluctuations on the microsecond-to-millisecond timescale that occur in ScCM. In the absence of allosteric effectors, ScCM did not exclusively exchange between T and R conformations, suggesting that the two-state MWC model is insufficient to explain conformational dynamics. Addition of tyrosine led to the quenching of much of the motion on this timescale, while new motions were identified in the presence of tryptophan. These new motions are consistent with conformational fluctuations into an alternative conformation that may be important for enzyme activity.
Sujet(s)
Chorismate mutase , Protéines de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Régulation allostérique , Chorismate mutase/composition chimique , Spectroscopie par résonance magnétique , Résonance magnétique nucléaire biomoléculaire , Conformation des protéines , Saccharomyces cerevisiae/enzymologie , Protéines de Saccharomyces cerevisiae/composition chimique , Tryptophane/métabolisme , Tyrosine/métabolismeRÉSUMÉ
The current theoretical perception of enzymatic activity is highly reliant on the determination of the activation energy of the reactions, which is often calculated using computationally demanding quantum mechanical calculations. With the ever-increasing use of bioengineering techniques that produce too many variants of the same enzyme, a fast and accurate way to study the relative efficiency of enzymes is currently in high demand. Here, we propose the local electric field (LEF) of the enzyme along the reaction axis as a descriptor for the enzymatic activity using the example of chorismate mutase in its native form and several variants (R90A, R90G, and R90K/C88S). The study shows a direct correlation between the calculated enzymatic EF and the enzymatic activity for all the complexes. MD simulations of the Michaelis complex and the transition state analog (TSA) show a stabilizing force on the TSA due to the enzymatic EF. QM/MM and QM-only DFT calculations in the presence of an external electric field (EEF) oriented along the reaction axis show that the electric field can interact with the dipole moment of the TS, thereby stabilizing it and thus lowering the activation energy.
Sujet(s)
Chorismate mutase/composition chimique , Biocatalyse , Chorismate mutase/génétique , Acide chorismique/composition chimique , Théorie de la fonctionnelle de la densité , Modèles chimiques , Simulation de dynamique moléculaire , Mutation , Électricité statique , ThermodynamiqueRÉSUMÉ
The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this pathway, intensified and revealed to be a promising strategy to develop new anticancer drugs. Among attractive PHGDH inhibitors are the α-ketothioamides. In previous work, we have demonstrated their efficacy in the inhibition of PHGDH in vitro and in cellulo. However, the precise site of action of this series, which would help the rational design of new inhibitors, remained undefined. In the present study, the detailed mechanism-of-action of a representative α-ketothioamide inhibitor is reported using several complementary experimental techniques. Strikingly, our work led to the identification of an allosteric site on PHGDH that can be targeted for drug development. Using mass spectrometry experiments and an original α-ketothioamide diazirine-based photoaffinity probe, we identified the 523Q-533F sequence on the ACT regulatory domain of PHGDH as the binding site of α-ketothioamides. Mutagenesis experiments further documented the specificity of our compound at this allosteric site. Our results thus pave the way for the development of new anticancer drugs using a completely novel mechanism-of-action.
Sujet(s)
Diazo-méthane/composition chimique , Antienzymes/pharmacologie , Spectrométrie de masse/méthodes , Phosphoglycerate dehydrogenase/antagonistes et inhibiteurs , Phosphoglycerate dehydrogenase/métabolisme , Site allostérique , Aspartate kinase/composition chimique , Aspartate kinase/métabolisme , Sites de fixation , Chorismate mutase/composition chimique , Chorismate mutase/métabolisme , Humains , Structure moléculaire , Domaines protéiques , Relation structure-activitéRÉSUMÉ
The stringent response, regulated by the bifunctional (p)ppGpp synthetase/hydrolase Rel in mycobacteria, is critical for long-term survival of the drug-tolerant dormant state of Mycobacterium tuberculosis. During amino acid starvation, MtRel senses a drop in amino acid concentration and synthesizes the messengers pppGpp and ppGpp, collectively called (p)ppGpp. Here, we investigate the role of the regulatory 'Aspartokinase, Chorismate mutase and TyrA' (ACT) domain in MtRel. Using NMR spectroscopy approaches, we report the high-resolution structure of dimeric MtRel ACT which selectively binds to valine out of all other branched-chain amino acids tested. A set of MtRel ACT mutants were generated to identify the residues required for maintaining the head-to-tail dimer. Through NMR titrations, we determined the crucial residues for binding of valine and show structural rearrangement of the MtRel ACT dimer in the presence of valine. This study suggests the direct involvement of amino acids in (p)ppGpp accumulation mediated by MtRel independent to interactions with stalled ribosomes. Database Structural data are available in the PDB database under the accession number 6LXG.
Sujet(s)
Aspartate kinase/génétique , Chorismate mutase/génétique , Ligases/génétique , Mycobacterium tuberculosis/génétique , Aspartate kinase/composition chimique , Aspartate kinase/ultrastructure , Chorismate mutase/composition chimique , Chorismate mutase/ultrastructure , Guanosine tétraphosphate/génétique , Hydrolases/génétique , Ligases/composition chimique , Ligases/ultrastructure , Spectroscopie par résonance magnétique , Mycobacterium tuberculosis/pathogénicité , Domaines protéiques/génétique , Multimérisation de protéines , Facteurs de transcription/génétiqueRÉSUMÉ
The rational design of enzymes is an important goal for both fundamental and practical reasons. Here, we describe a process to learn the constraints for specifying proteins purely from evolutionary sequence data, design and build libraries of synthetic genes, and test them for activity in vivo using a quantitative complementation assay. For chorismate mutase, a key enzyme in the biosynthesis of aromatic amino acids, we demonstrate the design of natural-like catalytic function with substantial sequence diversity. Further optimization focuses the generative model toward function in a specific genomic context. The data show that sequence-based statistical models suffice to specify proteins and provide access to an enormous space of functional sequences. This result provides a foundation for a general process for evolution-based design of artificial proteins.
Sujet(s)
Chorismate mutase , Évolution moléculaire , Modèles génétiques , Modèles statistiques , Séquence d'acides aminés , Chorismate mutase/composition chimique , Chorismate mutase/génétique , Protéines Escherichia coli/composition chimique , Protéines Escherichia coli/génétiqueRÉSUMÉ
Allosteric regulation is important in many biological processes, including cell signaling, gene regulation, and metabolism. Saccharomyces cerevisiae chorismate mutase (ScCM) is a key homodimeric enzyme in the shikimate pathway responsible for the generation of aromatic amino acids, where it is allosterically inhibited and activated by Tyr and Trp, respectively. Our previous studies indicated that binding of both allosteric effectors is negatively cooperative, that is binding at one allosteric binding site discourages binding at the other, due to the entropic penalty of binding the second allosteric effector. We utilized variable temperature isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) experiments to better understand the entropic contributions to allosteric effector binding, including changes to solvent entropy and protein conformational entropy. Upon binding either Tyr or Trp, ScCM experiences a quenching of motions on the picosecond-to-nanosecond time scale, which we could relate to a loss of protein conformational entropy. Further ITC and NMR studies were consistent with the Tyr-bound form of ScCM being associated with more water molecules compared to the Trp-bound form and Tyr binding being associated with a less positive solvent entropy change. These studies provide insight into the role of structural dynamics in ScCM function and highlight the importance of solvent entropy changes in allosteric regulation, a historically underappreciated concept.
Sujet(s)
Chorismate mutase/composition chimique , Chorismate mutase/métabolisme , Entropie , Saccharomyces cerevisiae/enzymologie , Solvants/composition chimique , Tryptophane/composition chimique , Tyrosine/composition chimique , Régulation allostérique , Site allostérique , Acides aminés aromatiques/métabolisme , Cristallographie aux rayons X/méthodes , Modèles moléculaires , Conformation des protéines , Multimérisation de protéines , Tryptophane/métabolisme , Tyrosine/métabolismeRÉSUMÉ
Amino acid metabolic enzymes often contain a regulatory ACT domain, named for aspartate kinase, chorismate mutase, and TyrA (prephenate dehydrogenase). Arabidopsis encodes 12 putative amino acid sensor ACT repeat (ACR) proteins, all containing ACT repeats but no identifiable catalytic domain. Arabidopsis ACRs comprise three groups based on domain composition and sequence: group I and II ACRs contain four ACTs each, and group III ACRs contain two ACTs. Previously, all three groups had been documented only in Arabidopsis. Here, we extended this to algae and land plants, showing that all three groups of ACRs are present in most, if not all, land plants, whereas among algal ACRs, although quite diverse, only group III is conserved. The appearance of canonical group I and II ACRs thus accompanied the evolution of plants from living in water to living on land. Alignment of ACTs from plant ACRs revealed a conserved motif, DRPGLL, at the putative ligand-binding site. Notably, the unique features of the DRPGLL motifs in each ACT domain are conserved in ACRs from algae to land plants. The conservation of plant ACRs is reminiscent of that of human cellular arginine sensor for mTORC1 (CASTOR1), a member of a small protein family highly conserved in animals. CASTOR proteins also have four ACT domains, although the sequence identities between ACRs and CASTORs are very low. Thus, plant ACRs and animal CASTORs may have adapted the regulatory ACT domains from a more ancient metabolic enzyme, and then evolved independently.
Sujet(s)
Acides aminés/métabolisme , Aspartate kinase/classification , Chorismate mutase/classification , Évolution moléculaire , Oryza/enzymologie , Protéines végétales/classification , Prephenate dehydrogenase/classification , Motifs d'acides aminés , Arabidopsis/enzymologie , Aspartate kinase/composition chimique , Chlorophyta/enzymologie , Chorismate mutase/composition chimique , Séquence conservée , Phylogenèse , Protéines végétales/composition chimique , Prephenate dehydrogenase/composition chimique , Domaines protéiques , Rhodophyta/enzymologieRÉSUMÉ
Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105m-1s-1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.
Sujet(s)
Protéines bactériennes/métabolisme , Chorismate mutase/métabolisme , Mycobacterium tuberculosis/enzymologie , 3-Deoxy-7-phosphoheptulonate synthase/composition chimique , 3-Deoxy-7-phosphoheptulonate synthase/métabolisme , Régulation allostérique , Séquence d'acides aminés , Protéines bactériennes/composition chimique , Protéines bactériennes/génétique , Biocatalyse , Domaine catalytique , Chorismate mutase/composition chimique , Chorismate mutase/génétique , Cristallographie aux rayons X , Évolution moléculaire dirigée , Cinétique , Simulation de dynamique moléculaire , Mutagenèse , Structure quaternaire des protéines , Protéines recombinantes/biosynthèse , Protéines recombinantes/composition chimique , Alignement de séquences , Acide shikimique/métabolisme , Température de transitionRÉSUMÉ
In solution, proteins fluctuate among many conformational substates, with their relative free energies determining substate populations and energy barriers determining conformational exchange kinetics. It has been suggested that members of the conformational ensemble may be responsible for different protein functions, although it is generally difficult to test such a proposal in most systems. A model protein for deciphering individual substate contributions is the homodimeric Saccharomyces cerevisiae chorismate mutase (ScCM) enzyme, which is negatively and positively regulated by tyrosine and tryptophan, respectively. Previous X-ray crystallography structures revealed two equivalent allosteric binding pockets that can be occupied by either tryptophan or tyrosine. We proposed that under cellular conditions there are six potential states of ScCM: no allosteric effector bound, a single tyrosine bound, a single tryptophan bound, two tyrosines bound, two tryptophans bound, and a mixed bound state in which tyrosine and tryptophan occupy different allosteric sites. We used isothermal titration calorimetry and solution-state nuclear magnetic resonance spectroscopy to confirm the existence of all six states and construct the complete six-state equilibrium binding profile. We were also able to assign enzyme activities to each state, which allowed us to derive the enzyme activity landscape across the range of cellular concentrations of tyrosine and tryptophan. Surprisingly, the mixed bound state had the highest enzyme activity, which suggested that the shikimate pathway is shunted toward tyrosine production under most conditions.
Sujet(s)
Site allostérique , Chorismate mutase/composition chimique , Chorismate mutase/métabolisme , Saccharomyces cerevisiae/enzymologie , Tryptophane/métabolisme , Tyrosine/métabolisme , Régulation allostérique , Calorimétrie différentielle à balayage , Domaine catalytique , Dimérisation , Activation enzymatique , Escherichia coli/génétique , Cinétique , Spectroscopie par résonance magnétique , Modèles moléculaires , Structure secondaire des protéines , Tryptophane/composition chimique , Tyrosine/composition chimiqueRÉSUMÉ
Combined quantum mechanics/molecular mechanics (QM/MM) methods are increasingly widely utilized in studies of reactions in enzymes and other large systems. Here, we apply a range of QM/MM methods to investigate the Claisen rearrangement of chorismate to prephenate, in solution, and in the enzyme chorismate mutase. Using projector-based embedding in a QM/MM framework, we apply treatments up to the CCSD(T) level. We test a range of density functional QM/MM methods and QM region sizes. The results show that the calculated reaction energetics are significantly more sensitive to the choice of density functional than they are to the size of the QM region in these systems. Projector-based embedding of a wave function method in DFT reduced the 13 kcal/mol spread in barrier heights calculated at the DFT/MM level to a spread of just 0.3 kcal/mol, essentially eliminating dependence on the functional. Projector-based embedding of correlated ab initio methods provides a practical method for achieving high accuracy for energy profiles derived from DFT and DFT/MM calculations for reactions in condensed phases.
Sujet(s)
Théorie de la fonctionnelle de la densité , Enzymes/composition chimique , Domaine catalytique , Chorismate mutase/composition chimique , Chorismate mutase/métabolisme , Enzymes/métabolisme , Modèles moléculaires , ThermodynamiqueRÉSUMÉ
Fungi-induced plant diseases affect global food security and plant ecology. The biotrophic fungus Ustilago maydis causes smut disease in maize (Zea mays) plants by secreting numerous virulence effectors that reprogram plant metabolism and immune responses1,2. The secreted fungal chorismate mutase Cmu1 presumably affects biosynthesis of the plant immune signal salicylic acid by channelling chorismate into the phenylpropanoid pathway3. Here we show that one of the 20 maize-encoded kiwellins (ZmKWL1) specifically blocks the catalytic activity of Cmu1. ZmKWL1 hinders substrate access to the active site of Cmu1 through intimate interactions involving structural features that are specific to fungal Cmu1 orthologues. Phylogenetic analysis suggests that plant kiwellins have a versatile scaffold that can specifically counteract pathogen effectors such as Cmu1. We reveal the biological activity of a member of the kiwellin family, a widely conserved group of proteins that have previously been recognized only as important human allergens.
Sujet(s)
Antigènes végétaux/métabolisme , Maladies des plantes/microbiologie , Ustilago/métabolisme , Ustilago/pathogénicité , Facteurs de virulence/métabolisme , Zea mays/métabolisme , Zea mays/microbiologie , Chorismate mutase/antagonistes et inhibiteurs , Chorismate mutase/composition chimique , Chorismate mutase/métabolisme , Acide chorismique/métabolisme , Modèles moléculaires , Phylogenèse , Maladies des plantes/immunologie , Acide salicylique/immunologie , Ustilago/enzymologie , Zea mays/immunologieRÉSUMÉ
The Claisen rearrangement is a carbon-carbon bond-forming, pericyclic reaction of fundamental importance due to its relevance in synthetic and mechanistic investigations of organic and biological chemistry. Despite continued efforts, the molecular origins of the rate acceleration in going from the aqueous phase into the protein is still incompletely understood. In the present work, the rearrangement reactions for allyl-vinyl-ether (AVE), its dicarboxylated variant (AVE-(CO2)2), and the biologically relevant substrate chorismate are investigated in the gas phase, water, and in chorismate mutase. Only the rearrangement of chorismate in the enzyme shows a negative differential barrier when compared to the reaction in water, which leads to the experimentally observed catalytic effect for the enzyme. The molecular origin of this effect is the positioning of AVE-(CO2)2 and chorismate in the protein active site compared to AVE. Furthermore, in going from AVE-(CO2)2 to chorismate, entropic effects due to rigidification and ring formation are operative, which lead to changes in the rate. On the basis of "More O'Ferrall-Jencks" diagrams, it is confirmed that C-O bond breaking precedes C-C bond formation in all cases. This effect becomes more pronounced in going from the gas phase to the protein.
Sujet(s)
Acide chorismique/composition chimique , Éthers/composition chimique , Composés allyliques/composition chimique , Bacillus subtilis/enzymologie , Chorismate mutase/composition chimique , Simulation de dynamique moléculaire , Phénomènes de chimie organique , Thermodynamique , Composés vinyliques/composition chimiqueRÉSUMÉ
Fungal infections are spreading widely worldwide, and the types of treatment are limited due to the lack of diverse therapeutic agents and their associated side effects and toxicity. The discovery of new antifungal classes is vital and critical. We discovered the antifungal activity of abscisic acid through a rational drug design methodology that included the building of homology models for fungal chorismate mutases and a pharmacophore model derived from a transition state inhibitor. Ligand-based virtual screening resulted in some hits that were filtered using molecular docking and molecular dynamic simulations studies. Both in silico methods and in vitro antifungal assays were used as tools to select and validate the abscisic acid repurposing. Abscisic acid inhibition assays confirmed the inhibitory effect of abscisic acid on chorismate mutase through the inhibition of phenylpyruvate production. The repositioning of abscisic acid, the well-known and naturally occurring plant growth regulator, as a potential antifungal agent because of its suggested action as an inhibitor to several fungal chorismate mutases was the main result of this work.
Sujet(s)
Acide abscissique/composition chimique , Antifongiques/composition chimique , Chorismate mutase , Antienzymes/composition chimique , Protéines fongiques , Simulation de dynamique moléculaire , Chorismate mutase/antagonistes et inhibiteurs , Chorismate mutase/composition chimique , Découverte de médicament , Protéines fongiques/antagonistes et inhibiteurs , Protéines fongiques/composition chimiqueRÉSUMÉ
Burkholderia thailandensis is often used as a model for more virulent members of this genus of proteobacteria that are highly antibiotic-resistant and are potential agents of biological warfare that are infective by inhalation. As part of ongoing efforts to identify potential targets for the development of rational therapeutics, the structures of enzymes that are absent in humans, including that of chorismate mutase from B. thailandensis, have been determined by the Seattle Structural Genomics Center for Infectious Disease. The high-resolution structure of chorismate mutase from B. thailandensis was determined in the monoclinic space group P21 with three homodimers per asymmetric unit. The overall structure of each protomer has the prototypical AroQγ topology and shares conserved binding-cavity residues with other chorismate mutases, including those with which it has no appreciable sequence identity.
Sujet(s)
Burkholderia/enzymologie , Burkholderia/génétique , Chorismate mutase/composition chimique , Chorismate mutase/génétique , Séquence d'acides aminés , Cristallisation/méthodes , Structure secondaire des protéinesRÉSUMÉ
The bacterium Burkholderia phymatum is a promiscuous symbiotic nitrogen-fixating bacterium that belongs to one of the largest groups of Betaproteobacteria. Other Burkholderia species are known to cause disease in plants and animals, and some are potential agents for biological warfare. Structural genomics efforts include characterizing the structures of enzymes from pathways that can be targeted for drug development. As part of these efforts, chorismate mutase from B. phymatum was produced and crystallized, and a 1.95â Å resolution structure is reported. This enzyme shares less than 33% sequence identity with other homologs of known structure. There are two classes of chorismate mutase: AroQ and AroH. The bacterial subclass AroQγ has reported roles in virulence. Chorismate mutase from B. phymatum has the prototypical AroQγ topology and retains the characteristic chorismate mutase active site. This suggests that substrate-based chorismate mutase inhibitors will not be specific and are likely to affect beneficial bacteria such as B. phymatum.
Sujet(s)
Burkholderiaceae/enzymologie , Chorismate mutase/composition chimique , Séquence d'acides aminés , Domaine catalytique , Cristallisation , Cristallographie aux rayons X , Modèles moléculaires , Conformation des protéines , Similitude de séquencesRÉSUMÉ
Corynebacterium glutamicum is widely used for the industrial production of amino acids, nucleotides, and vitamins. The shikimate pathway enzymes DAHP synthase (CgDS, Cg2391) and chorismate mutase (CgCM, Cgl0853) play a key role in the biosynthesis of aromatic compounds. Here we show that CgCM requires the formation of a complex with CgDS to achieve full activity, and that both CgCM and CgDS are feedback regulated by aromatic amino acids binding to CgDS. Kinetic analysis showed that Phe and Tyr inhibit CgCM activity by inter-enzyme allostery, whereas binding of Trp to CgDS strongly activates CgCM. Mechanistic insights were gained from crystal structures of the CgCM homodimer, tetrameric CgDS, and the heterooctameric CgCM-CgDS complex, refined to 1.1, 2.5, and 2.2 Å resolution, respectively. Structural details from the allosteric binding sites reveal that DAHP synthase is recruited as the dominant regulatory platform to control the shikimate pathway, similar to the corresponding enzyme complex from Mycobacterium tuberculosis.
Sujet(s)
3-Deoxy-7-phosphoheptulonate synthase/métabolisme , Chorismate mutase/métabolisme , Corynebacterium glutamicum/enzymologie , Tryptophane/métabolisme , Régulation allostérique , Acides aminés aromatiques/métabolisme , Chorismate mutase/composition chimique , Corynebacterium glutamicum/composition chimique , Corynebacterium glutamicum/métabolisme , Cristallographie aux rayons X , Activation enzymatique , Modèles moléculaires , Phénylalanine/métabolisme , Conformation des protéines , Multimérisation de protéines , Acide shikimique/métabolisme , Tyrosine/métabolismeRÉSUMÉ
Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plant chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0â Å X-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes.