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Allostery, where remote ligand binding alters protein function, is essential for the control of metabolism. Here, we have identified a highly sophisticated allosteric response that allows complex control of the pathway for aromatic amino acid biosynthesis in the pathogen Mycobacterium tuberculosis. This response is mediated by an enzyme complex for...
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... The inability to produce the soluble split *JbCDT domain precluded a similar set of measurements with the *JbCDTCM system. *AfCMCDT and *JbCDTCM are not feedback regulated by phenylalanine or tyrosine Intracellular CMs involved in biosynthetic pathways are known to be subject to feedback regulation (1-3) involving various features such as additional allosteric domains (59), dynamic dimer interfaces (16), or complexes with partner enzymes, which enable sophisticated inter-enzyme allostery (10,17,18,(60)(61)(62)(63)(64). In contrast, no feedback inhibition has been detected for the exported enzymes *MtCM (19) or *PaCDT (31,32,65). ...
Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of l-phenylalanine (Phe). Here, we report the discovery of novel and extremely rare bifunctional fusion enzymes, consisting of fused CM and CDT domains, which are exported from the cytoplasm. Such enzymes were found in only nine bacterial species belonging to non-pathogenic γ- or β-Proteobacteria. In γ-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas in β-Proteobacteria the order is inverted. The CM domains share 15-20% sequence identity with the AroQγ class CM holotype of Mycobacterium tuberculosis (*MtCM), and the CDT domains 40-60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). In vitro kinetics revealed a Km <7 μM, much lower than for *MtCM, whereas kinetic parameters are similar for CDT domains and PheC. There is no feedback inhibition of CM or CDT by the pathway's end product Phe, and no catalytic benefit of the domain fusion compared to engineered single-domain constructs. The fusion enzymes of Aequoribacter fuscus, Janthinobacterium sp. HH01, and Duganella sacchari were crystallized and their structures refined to 1.6, 1.7, and 2.4 Å resolution, respectively. Neither the crystal structures nor size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for cooperation of CM and CDT active sites. The genetic neighborhood with genes encoding transporter and substrate binding proteins suggests that these exported bifunctional fusion enzymes may participate in signaling systems rather than in the biosynthesis of Phe.
... Intracellular CMs involved in biosynthetic pathways are known to be subject to feedback regulation 1-3 involving various features such as additional allosteric domains, 57 dynamic dimer interfaces, 16 or complexes with partner enzymes, which enable sophisticated inter-enzyme allostery. 10, 17,18,[58][59][60][61][62] In contrast, no feedback inhibition has been detected for the exported enzymes *MtCM 19 or *PaCDT. 63 To test whether *AfCMCDT and *JbCDTCM are subject to feedback control, a coupled CM+CDT assay was performed in the presence of a large excess of either Phe or Tyr, the relevant end products of the shikimate pathway. ...
... 1,2 Moreover, they are not subjected to feedback regulation by the metabolic end products Phe or Tyr, which is otherwise ubiquitous in intracellular shikimate pathway enzymes. 1-3,10, [16][17][18][57][58][59][60] The particularly low Km values for the CM domains, which allow efficient binding of very low concentrations of the substrate chorismate, may hint at their true biological task. The genomic localization together with genes for solute-binding and transport . ...
Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of phenylalanine. Surprisingly, exported CMs and CDTs exist in bacterial pathogens. Here, we report the discovery of novel and extremely rare exported bifunctional fusion enzymes, consisting of fused CM and CDT domains. Such enzymes were found in only nine bacterial species belonging to non-pathogenic γ- or β-proteobacteria. In γ-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas in β-proteobacteria the order is inversed. The CM domains share 15-20% sequence identity with the AroQ γ class CM holotype of Mycobacterium tuberculosis (*MtCM), and the CDT domains 40-60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). In vitro kinetics revealed a K m <7 µM, much lower than for *MtCM, whereas kinetic parameters are similar for CDT domains and PheC. There is no feedback inhibition of CM or CDT by the pathway’s end product Phe, and no catalytic benefit of the domain fusion compared to engineered single-domain constructs. The fusion enzymes of Aequoribacter fuscus , Janthinobacterium sp. HH01, and Duganella sacchari were crystallized and their structures refined to 1.6, 1.7, and 2.4 Å resolution, respectively. Neither the crystal structures nor size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for cooperation of CM and CDT active sites. The genetic neighborhood with genes encoding transporter and substrate binding proteins suggests that these exported bifunctional fusion enzymes may participate in signaling systems rather than in the biosynthesis of Phe.
... 26 Complex formation also endows MtCM with feedback regulation by Tyr and Phe through binding of these effectors to the MtDS partner. 21,27,28 Such inter-enzyme allosteric regulation 28 allows for dynamic adjustment of the CM activity to meet the changing needs of the cell. ...
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.
... This interaction leads to a rearrangement of active site residues to catalytically more favorable conformations (Fig. 1B) (21) and is likely a key contributing factor for the increase in CM activity, as shown by randomizing mutagenesis of the Cterminus followed by selection for functional variants (26). Complex formation also endows MtCM with feedback regulation by Tyr and Phe through binding of these effectors to the MtDS partner (21,27,28). Such inter-enzyme allosteric regulation (28) allows for dynamic adjustment of the CM activity to meet the changing needs of the cell. ...
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 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 pre-organized 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 MD simulations and confirmed by kinetic investigations of engineered variants.
... A similar functional reliance between the DAH7PS and CM is observed in the noncovalent DAH7PS-CM complex found in Corynebacterium glutamicum (CglDAH7PS-CM) (28) and Mycobacterium tuberculosis (MtuDAH7PS-CM) (29). In the MtuDAH7PS-CM complex, CM is activated upon complexation with DAH7PS and inhibited by the binding of allosteric inhibitors, Tyr and Phe, at allosteric binding sites within each DAH7PS subunit (30)(31)(32). The structure of the MtuDAH7PS-CM complex showed that the DAH7PS and CM subunits interact with each other extensively and form a stable interface ( Figure 1B) (29,31). ...
Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bi-enzyme 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 inter-reliance 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, loopC320-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.
... 128 Additionally, inter-enzyme allostery has been reported for chorismate mutase, e.g. in M. tuberculosis and Corynebacterium glutamicum where Trp-bound 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase binds to the inactive chorismate mutase, with the resulting complex increasing chorismate mutase activity thereby directing additional flux into the biosynthesis of aromatic amino acids. 54,129,130 In MST enzymes, allosteric regulation has been described for the glutamine amidotransferase domain of ADC synthases. Here, the formation of the nucleophile ammonia is allosterically activated by chorismate, binding in the active site. ...
Chorismate and isochorismate represent an important branching point connecting primary and secondary metabolism in bacteria, fungi, archaea and plants. Chorismate- and isochorismate-converting enzymes are potential targets for new bioactive compounds, as well as valuable biocatalysts for the in vivo and in vitro synthesis of fine chemicals. The diversity of the products of chorismate- and isochorismate-converting enzymes is reflected in the enzymatic three-dimensional structures and molecular mechanisms. Due to the high reactivity of chorismate and its derivatives, these enzymes have evolved to be accurately tailored to their respective reaction; at the same time, many of them exhibit a fascinating flexibility regarding side reactions and acceptance of alternative substrates. Here, we give an overview of the different (sub)families of chorismate- and isochorismate-converting enzymes, their molecular mechanisms, and three-dimensional structures. In addition, we highlight important results of mutagenetic approaches that generate a broader understanding of the influence of distinct active site residues for product formation and the conversion of one subfamily into another. Based on this, we discuss to what extent the recent advances in the field might influence the general mechanistic understanding of chorismate- and isochorismate-converting enzymes. Recent discoveries of new chorismate-derived products and pathways, as well as biocatalytic conversions of non-physiological substrates, highlight how this vast field is expected to continue developing in the future.
... In the branched pathway leading to the production of Trp, anthranilate synthase [9][10][11][12][13][14] and anthranilate phosphoribosyltransferase [15][16][17] have been reported with feedback regulation by Trp. In the Phe/Tyr biosynthesis branch, chorismate mutase [18][19][20], prephenate dehydratase [21][22][23][24] and prephenate dehydrogenase [25,26] were known to have feedback regulation. ...
Allostery, in which binding of ligands to remote sites causes a functional change in the active sites, is a fascinating phenomenon observed in enzymes. Allostery can occur either with or without significant conformational changes in the enzymes, and the molecular basis of its mechanism can be difficult to decipher using only experimental techniques. Computational tools for analyzing enzyme sequences, structures, and dynamics can provide insights into the allosteric mechanism at the atomic level. Combining computational and experimental methods offers a powerful strategy for the study of enzyme allostery. The aromatic amino acid biosynthesis pathway is essential in microorganisms and plants. Multiple enzymes involved in this pathway are sensitive to feedback regulation by pathway end products and are known to use allostery to control their activities. To date, four enzymes in the aromatic amino acid biosynthesis pathway have been computationally investigated for their allosteric mechanisms, including 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, anthranilate synthase, chorismate mutase, and tryptophan synthase. Here we review the computational studies and findings on the allosteric mechanisms of these four enzymes. Results from these studies demonstrate the capability of computational tools and encourage future computational investigations of allostery in other enzymes of this pathway.
... In fact, the poor activity of the MtCM dimer is essential for effective shikimate pathway regulation, exerted through inter-enzyme allostery in M. tuberculosis. We (1,11) and others (22,23) have shown that binding of the allosteric feedback inhibitors Phe and Tyr to MtDS induces MtCM-MtDS complex dissociation and thereby a shift from high to low intracellular CM activity, providing tight control over cytoplasmic aromatic amino acid concentrations. ...
... At the same time, the evolution experiment demonstrated that the compromised natural enzyme MtCM already possesses all functional groups required for efficient catalysis despite lacking otherwise absolutely conserved active site residues. The relatively facile evolutionary trajectory to boost activity by 2-3 orders of magnitude implies that AroQδ sub-class CMs on their own had evolved to be intentionally poor natural catalysts for enabling interenzyme allosteric regulation by switching between mediocre and highly active states (1,2,11,22,23). We expect that analogous rigorous evolutionary studies of natural enzymes will elucidate capabilities and limitations of numerous biocatalysts and shed light on yet undiscovered allosteric control mechanisms. Furthermore, the ease of finding beneficial mutations gives fundamental insights into the development of drug resistance and the evolution of enzyme function. ...
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 DAHP 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 k cat / K m 270-fold to 5 × 10 ⁵ M ⁻¹ s ⁻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 to 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.
... It has been shown that the allosteric machinery of MtuDAH7PS can be utilized by another small protein partner, chorismate mutase (MtuCM), by forming a noncovalent complex. [16][17][18] CM catalyzes a downstream reaction in the shikimate pathway, converting chorismate to prephenate, and is at the start of the branchpoint of the shikimate pathway that leads to the production of Phe and Tyr. It has been shown that complex formation between MtuDAH7PS and MtuCM significantly enhances the catalytic activity of MtuCM. ...
... It has been shown that complex formation between MtuDAH7PS and MtuCM significantly enhances the catalytic activity of MtuCM. [16][17][18] Furthermore, the normally unregulated MtuCM becomes sensitive to inhibition by Phe, which binds to the dimer interface on MtuDAH7PS. [16][17][18] A similar scenario was also observed in another type II enzyme, i.e. the DAH7PS from Corynebacterium glutamicum, 19 indicating this inter-enzyme communication may be a more common feature among the type II DAH7PS enzymes. ...
... [16][17][18] Furthermore, the normally unregulated MtuCM becomes sensitive to inhibition by Phe, which binds to the dimer interface on MtuDAH7PS. [16][17][18] A similar scenario was also observed in another type II enzyme, i.e. the DAH7PS from Corynebacterium glutamicum, 19 indicating this inter-enzyme communication may be a more common feature among the type II DAH7PS enzymes. communication networks that allow for such sophisticated allosteric control of the gatekeeper enzyme for aromatic biosynthesis. ...
Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles towards functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-D -arabino -heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis , which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branch-point enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkedly, generating a non-allosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.
... Furthermore, MtDAHPS also forms a non-covalent hetero-octameric complex with M. tuberculosis chorismate mutase (MtCM), which acts at the branch point that connects the shikimate pathway to Phe and Tyr production [26,37,38]. This complex formation not only activates MtCM activity by more than two orders of magnitude but also allows MtCM to share the allosteric machinery located on MtDAHPS to direct the shikimate pathway end product-chorismate-towards either Trp or Phe and Tyr biosynthesis, based on metabolic requirements [39,40]. Interestingly, this remote control between DAHPS (the first enzyme of the shikimate pathway) and chorismate mutase represents a novel paradigm for regulation of this pathway [38]. ...
... tuberculosis chorismate mutase (MtCM), which acts at the branch point that connects the shikimate pathway to Phe and Tyr production [26,37,38]. This complex formation not only activates MtCM activity by more than two orders of magnitude but also allows MtCM to share the allosteric machinery located on MtDAHPS to direct the shikimate pathway end product-chorismatetowards either Trp or Phe and Tyr biosynthesis, based on metabolic requirements [39,40]. Interestingly, this remote control between DAHPS (the first enzyme of the shikimate pathway) and chorismate mutase represents a novel paradigm for regulation of this pathway [38]. ...
Roughly a third of the world’s population is estimated to have latent Mycobacterium tuberculosis infection, being at risk of developing active tuberculosis (TB) during their lifetime. Given the inefficacy of prophylactic measures and the increase of drug-resistant M. tuberculosis strains, there is a clear and urgent need for the development of new and more efficient chemotherapeutic agents, with selective toxicity, to be implemented on patient treatment. The component enzymes of the shikimate pathway, which is essential in mycobacteria and absent in humans, stand as attractive and potential targets for the development of new drugs to treat TB. This review gives an update on published work on the enzymes of the shikimate pathway and some insight on what can be potentially explored towards selective drug development.
