Role of Half-of-Sites Reactivity and Inter-Subunit Communications in DAHP Synthase Catalysis and Regulation.

α-Carboxyketose synthases, including 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS), are long-standing targets for inhibition. They are challenging targets to create tight-binding inhibitors against, and inhibitors often display half-of-sites binding and partial inhibition. Half-of-sites inhibition demonstrates the existence of inter-subunit communication in DAHPS. We used X-ray crystallography and spatially resolved hydrogen-deuterium exchange (HDX) to reveal the structural and dynamic bases for inter-subunit communication in Escherichia coli DAHPS(Phe), the isozyme that is feedback-inhibited by phenylalanine. Crystal structures of this homotetrameric (dimer-of-dimers) enzyme are invariant over 91% of its sequence. Three variable loops make up 8% of the sequence and are all involved in inter-subunit contacts across the tight-dimer interface. The structures have pseudo-twofold symmetry indicative of inter-subunit communication across the loose-dimer interface, with the diagonal subunits B and C always having the same conformation as each other, while subunits A and D are variable. Spatially resolved HDX reveals contrasting responses to ligand binding, which, in turn, affect binding of the second substrate, erythrose-4-phosphate (E4P). The N-terminal peptide, M1-E12, and the active site loop that binds E4P, F95-K105, are key parts of the communication network. Inter-subunit communication appears to have a catalytic role in all α-carboxyketose synthase families and a regulatory role in some members.

Protein engineering for feedback resistance in 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

The shikimate pathway delivers aromatic amino acids (AAAs) in prokaryotes, fungi, and plants and is highly utilized in the industrial synthesis of bioactive compounds. Carbon flow into this pathway is controlled by the initial enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS). AAAs produced further downstream, phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), regulate DAHPS by feedback inhibition. Corynebacterium glutamicum, the industrial workhorse for amino acid production, has two isoenzymes of DAHPS, AroF (Tyr sensitive) and AroG (Phe and Tyr sensitive). Here, we introduce feedback resistance against Tyr in the class I DAHPS AroF (AroFcg). We pursued a consensus approach by drawing on structural modeling, sequence and structural comparisons, knowledge of feedback-resistant variants in E. coli homologs, and computed folding free energy changes. Two types of variants were predicted: Those where substitutions putatively either destabilize the inhibitor binding site or directly interfere with inhibitor binding. The recombinant variants were purified and assessed in enzyme activity assays in the presence or absence of Tyr. Of eight AroFcg variants, two yielded > 80% (E154N) and > 50% (P155L) residual activity at 5 mM Tyr and showed > 50% specific activity of the wt AroFcg in the absence of Tyr. Evaluation of two and four further variants at positions 154 and 155 yielded E154S, completely resistant to 5 mM Tyr, and P155I, which behaves similarly to P155L. Hence, feedback-resistant variants were found that are unlikely to evolve by point mutations from the parental gene and, thus, would be missed by classical strain engineering. KEY POINTS: • We introduce feedback resistance against Tyr in the class I DAHPS AroF • Variants at position 154 (155) yield > 80% (> 50%) residual activity at 5 mM Tyr • The variants found are unlikely to evolve by point mutations from the parental gene.

Evaluation of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS) as a Vulnerable Target in Mycobacterium tuberculosis.

Tuberculosis (TB) remains one of the leading causes of death due to a single pathogen. The emergence and proliferation of multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) represent compelling reasons to invest in the pursuit of new anti-TB agents. The shikimate pathway, responsible for chorismate biosynthesis, which is a precursor of important aromatic compounds, is required for Mycobacterium tuberculosis growth. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (MtbDAHPS) catalyzes the first step in the shikimate pathway and it is an attractive target for anti-tubercular agents. Here, we used a CRISPRi system to evaluate the DAHPS as a vulnerable target in M. tuberculosis. The silencing of aroG significantly reduces the M. tuberculosis growth in both rich medium and, especially, in infected murine macrophages. The supplementation with amino acids was only able to partially rescue the growth of bacilli, whereas the Aro supplement (aromix) was enough to sustain the bacterial growth at lower rates. This study shows that MtbDAHPS protein is vulnerable and, therefore, an attractive target to develop new anti-TB agents. In addition, the study contributes to a better understanding of the biosynthesis of aromatic compounds and the bacillus physiology. IMPORTANCE Determining the vulnerability of a potential target allows us to assess whether its partial inhibition will impact bacterial growth. Here, we evaluated the vulnerability of the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) from M. tuberculosis by silencing the DAHPS-coding aroG gene in different contexts. These results could lead to the development of novel and potent anti-tubercular agents in the near future.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase as the gatekeeper of plant aromatic natural product biosynthesis.

The shikimate pathway connects the central carbon metabolism with the biosynthesis of aromatic amino acids-l-tyrosine, l-phenylalanine, and l-tryptophan-which play indispensable roles as precursors of numerous aromatic phytochemicals. Despite the importance of the shikimate pathway-derived products for both plant physiology and human society, the regulatory mechanism of the shikimate pathway remains elusive. This review summarizes the recent progress and current understanding on the plant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHP synthase or DHS) enzymes that catalyze the committed reaction of the shikimate pathway. We particularly focus on how the DHS activity is regulated in plants in comparison to those of microbes and discuss potential roles of DHS as the critical gatekeeper for the production of plant aromatic compounds.

Elevated tyrosine results in the cytosolic retention of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in Arabidopsis thaliana.

The shikimate pathway plays a central role in the biosynthesis of aromatic amino acids and specialized metabolites in plants. The first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) serves as a key regulatory point for the pathway in various organisms. These enzymes are important in regulating the shikimate pathway in multiple microbial systems. The mechanism of regulation of DAHPS is poorly understood in plants, and the role of tyrosine (Tyr) with respect to the three DAHPS isozymes from Arabidopsis thaliana was investigated. In vitro enzymatic analyses established that Tyr does not function as an allosteric regulator for the A. thaliana DAHPS isozymes. In contrast, Arabidopsis T-DNA insertional mutants for the DAHPS1 locus, dahps1, are hypersensitive to elevated Tyr. Tyr hypersensitivity can be reversed with tryptophan and phenylalanine supplementation, indicating that Tyr is affecting the shikimate pathway flux in the dahps1 mutant. Tyr treatment of Arabidopsis seedlings showed reduced accumulation of overexpressed DAHPS2 in the chloroplast. Further, bimolecular fluorescence complementation studies revealed that DAHPS2 interacts with a 14-3-3 protein in the cytosol, and this interaction is enhanced with Tyr treatment. This interaction with 14-3-3 may retain DAHPS2 in the cytosol, which prevents its ability to function in the chloroplast with elevated Tyr.

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, 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.

Obtainment, selection and characterization of a mutant strain of Kluyveromyces marxianus that displays improved production of 2-phenylethanol and enhanced DAHP synthase activity.

AIMS: Yeasts produce 2-phenylethanol (2-PE) from sugars via de novo synthesis; however, its synthesis is limited due to feedback inhibition on the isofunctional 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthases (Aro3p and Aro4p). This work aimed to select Kluyveromyces marxianus mutant strains with improved capacity to produce 2-PE from sugars. METHODS AND RESULTS: Kluyveromyces marxianus CCT 7735 mutant strains were selected from UV irradiation coupled with screening of p-fluoro-dl-phenylalanine (PFP) tolerant strains on culture medium without l-Phe addition. Most of them produced 2-PE titres higher than the parental strain and the Km_PFP41 mutant strain stood out for displaying the highest 2-PE specific production rate. Moreover it showed higher activity of DAHP synthase than the parental strain. We sequenced both ARO3 and ARO4 genes of Km_PFP41 mutant and identified mutations in ARO4 which caused changes in both size and conformation of the Aro4p. These changes seem to be associated with the enhanced activity of DAHP synthase and improved production of 2-PE exhibited by that mutant strain. CONCLUSIONS: The Km_PFP41 mutant strain presented improved 2-PE production via de novo synthesis and enhanced DAHP synthase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant strain obtained in this work may be exploited as a yeast cell factory for high-level synthesis of 2-PE.

Diverse allosteric componentry and mechanisms control entry into aromatic metabolite biosynthesis.

Allosteric regulation of the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) controls the entry into aromatic amino acid biosynthesis in plants and microorganisms. DAH7PS has acquired a diverse range of allosteric machinery to enable this functionality. This review provides an overview of the current knowledge of the structural basis of allostery in this enzyme family and the evolutionary relationships between the different solutions to allosteric control of aromatic metabolite biosynthesis.

A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in Mycobacterium tuberculosis.

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward 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 branchpoint 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 remarkably, generating a nonallosteric 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.

Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance.

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.

NeuNAc Oxime: A Slow-Binding and Effectively Irreversible Inhibitor of the Sialic Acid Synthase NeuB.

NeuB is a bacterial sialic acid synthase used by neuroinvasive bacteria to synthesize N-acetylneuraminate (NeuNAc), helping them to evade the host immune system. NeuNAc oxime is a potent slow-binding NeuB inhibitor. It dissociated too slowly to be detected experimentally, with initial estimates of its residence time in the active site being >47 days. This is longer than the lifetime of a typical bacterial cell, meaning that inhibition is effectively irreversible. Inhibition data fitted well to a model that included a pre-equilibration step with a Ki of 36 µM, followed by effectively irreversible conversion to an E*·I complex, with a k2 of 5.6 × 10-5 s-1. Thus, the inhibitor can subvert ligand release and achieve extraordinary residence times in spite of a relatively modest initial dissociation constant. The crystal structure showed the oxime functional group occupying the phosphate-binding site normally occupied by the substrate PEP and the tetrahedral intermediate. There was an ≈10% residual rate at high inhibitor concentrations regardless of how long NeuB and NeuNAc oxime were preincubated together. However, complete inhibition was achieved by incubating NeuNAc oxime with the actively catalyzing enzyme. This requirement for the enzyme to be actively turning over for the inhibitor to bind to the second subunit demonstrated an important role for intersubunit communication in the inhibitory mechanism.

Molecular basis for feedback inhibition of tyrosine-regulated 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) is responsible for the biosynthesis of essential aromatic compounds in microorganisms and plants. It plays a crucial role in the regulation of the carbon flow into the shikimate pathway. Until now, the crystal structures and regulatory mechanisms of dimeric DAHPS enzymes from type Iα subclass have not been reported. Here, we reported dimeric structures of the tyrosine-regulated DAHPS from Escherichia coli, both in its apo form and complex with the inhibitor tyrosine at 2.5 and 2.0 Šresolutions, respectively. DAHPS(Tyr) has a typical (ß/α)8 TIM barrel, which is decorated with an N-terminal extension and an antiparallel ß sheet, ß6a/ß6b. Inhibitor tyrosine binds at a cavity formed by residues of helices α3, α4, strands ß6a, ß6b and the adjacent loops, and directly interacts with residues P148, Q152, S181, I213 and N8*. Although the small angle X-ray scattering profiles from DAHPS(Tyr) with and without tyrosine shows that tyrosine binding leaves most of DAHPS(Tyr) structures unaffected. The comparison of the liganded and unliganded crystal structures reveals that conformational changes of residues P148, Q152 and I213 initiate a transmission pathway to propagate the allosteric signal from the tyrosine-binding site to the active site, which is different from DAHPS(Phe), a phenylalanine-regulated isozyme from E. coli. In addition, mutations of five tyrosine-binding residues P148, Q152, S181, I213 and N8* leads to tyrosine-resistant DAHPS(Tyr) enzymes. These findings provide a new insight into the regulatory mechanism of DAHPS enzymes and a basis for further engineering studies.

Eliminating Competition: Characterizing and Eliminating Competitive Binding at Separate Sites between DAHP Synthase's Essential Metal Ion and the Inhibitor DAHP Oxime.

3-Deoxy-d- arabinoheptulosonate 7-phosphate (DAHP) oxime is a transition state mimic inhibitor of bacterial DAHP synthase, with K i = 1.5 µM and a residence time of tR = 83 min. Unexpectedly, DAHP oxime inhibition is competitive with respect to the essential metal ion, Mn2+, even though the inhibitor and metal ion do not occupy the same physical space in the active site. This is problematic because DAHP synthase is activated by multiple divalent metal cations, some of which have significant intracellular concentrations and some of which dissociate slowly. The nature of DAHP oxime's competition with the metal ion was investigated. Inhibition shifted from metal-competitive at physiological pH to metal-noncompetitive at pH > 8.7 in response to deprotonation of the Cys61 side chain. The modes of inhibition of DAHP synthase mutants and inhibitor fragments demonstrated that metal-competitive inhibition arose from interactions between Mn2+, DAHP oxime's O4 hydroxyl group, and the Cys61 and Asp326 side chains. The majority of potent DAHP synthase inhibitors in the literature possess a 4-hydroxyl group. Removing it could avoid metal-competitive inhibition and avoid them being outcompeted by metal ions in vivo.

Structural and functional characterisation of the entry point to pyocyanin biosynthesis in Pseudomonas aeruginosa defines a new 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase subclass.

In Pseudomonas aeruginosa (Pae), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α2a and α2b, relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type IIA and type IIB based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.

A Pseudoisostructural Type II DAH7PS Enzyme from Pseudomonas aeruginosa: Alternative Evolutionary Strategies to Control Shikimate Pathway Flux.

The shikimate pathway is responsible for the biosynthesis of key aromatic metabolites in microorganisms and plants. The enzyme 3-deoxy-d- arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the pathway and DAH7PSs are classified as either type I or type II. The DAH7PSs from Pseudomonas aeruginosa are of particular interest as open reading frames encoding four putative DAH7PS isoenzymes, two classified as type Iα and two classified as type II, have been identified. Here, the structure of a type II DAH7PS enzyme from P. aeruginosa (PAO1) has been determined at 1.54 Å resolution, in complex with its allosteric inhibitor tryptophan. Structural differences in the extra-barrel elements, when compared to other type II DAH7PS enzymes, directly relate to the formation of a distinct quaternary conformation with consequences for allosteric function and the control of flux to branching pathways. In contrast to the well-characterized Mycobacterium tuberculosis type II DAH7PS, which binds multiple allosteric inhibitors, this PaeDAH7PSPA2843 is observed to be modestly allosterically inhibited by a single aromatic amino acid, tryptophan. In addition, structures in complex with tyrosine or with no allosteric ligand bound were determined. These structures provide new insights into the linkages between the active and allosteric sites. With four putative DAH7PS enzymes, P. aeruginosa appears to have evolved control of shikimate pathway flux at the genetic level, rather than control by multiple allosteric effectors to a single type II DAH7PS, as in M. tuberculosis. Type II DAH7PSs, thus, appear to have a more varied evolutionary trajectory than previously indicated.

Exploring modular allostery via interchangeable regulatory domains.

Most proteins comprise two or more domains from a limited suite of protein families. These domains are often rearranged in various combinations through gene fusion events to evolve new protein functions, including the acquisition of protein allostery through the incorporation of regulatory domains. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of aromatic amino acid biosynthesis and displays a diverse range of allosteric mechanisms. DAH7PSs adopt a common architecture with a shared (ß/α)8 catalytic domain which can be attached to an ACT-like or a chorismate mutase regulatory domain that operates via distinct mechanisms. These respective domains confer allosteric regulation by controlling DAH7PS function in response to ligand Tyr or prephenate. Starting with contemporary DAH7PS proteins, two protein chimeras were created, with interchanged regulatory domains. Both engineered proteins were catalytically active and delivered new functional allostery with switched ligand specificity and allosteric mechanisms delivered by their nonhomologous regulatory domains. This interchangeability of protein domains represents an efficient method not only to engineer allostery in multidomain proteins but to create a new bifunctional enzyme.

In vitro metal catalyzed oxidative stress in DAH7PS: Methionine modification leads to structure destabilization and induce amorphous aggregation.

The first committed step of the shikimate pathway is catalyzed by a metalloenzyme 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), which exhibits vulnerability to the oxidative stress. DAH7PS undergoes inactivation in multiple ways in the presence of redox metal, H2O2, and superoxide. The molecular mechanism and susceptibility of its inactivation might differ in different organisms and are presently unclear. In the present work, we have cloned, expressed and purified a DAH7PS from Providencia alcalifaciens (PaDAH7PS). The oligomeric state and effect of redox metal treatment on its stability were analyzed through the size exclusion chromatography. The FTIR, MALDI-TOF/TOF-MS studies revealed that methionine residues were modified to methionine sulfoxide in PaDAH7PS. During oxidation, PaDAH7PS is altered into partially folded protein and unfolded states as determined by CD and Fluorescence studies. A significant loss in enzymatic activity of PaDAH7PS was determined and the formation of amorphous aggregates was visualized using AFM imaging and also confirmed by ThT binding based assay. This is the first report where we have shown a hexameric DAH7PS and the methionine residues of PaDAH7PS get oxidize in the presence of oxidative stress. The partially folded and unfolded oligomeric states with high ß-content of PaDAH7PS might be the critical precursors for aggregation.

Quaternary structure is an essential component that contributes to the sophisticated allosteric regulation mechanism in a key enzyme from Mycobacterium tuberculosis.

The first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), adopts a range of distinct allosteric regulation mechanisms in different organisms, related to different quaternary assemblies. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) is a homotetramer, with the allosteric sites in close proximity to the interfaces. Here we examine the importance of the quaternary structure on catalysis and regulation, by amino acid substitution targeting the tetramer interface of MtuDAH7PS. Using only single amino acid substitutions either in, or remote from the interface, two dimeric variants of MtuDAH7PS (MtuDAH7PSF227D and MtuDAH7PSG232P) were successfully generated. Both dimeric variants maintained activity due to the distance between the sites of amino acid substitution and the active sites, but attenuated catalytic efficiency was observed. Both dimeric variants showed significantly disrupted allosteric regulation with greatly impaired binding affinity for one of the allosteric ligands. Molecular dynamics simulations revealed changes in protein dynamics and average conformations in the dimeric variant caused by amino acid substitution remote to the tetramer interface (MtuDAH7PSG232P), which are consistent with the observed reduction in catalytic efficiency and loss of allosteric response.

Structure of Chorismate Mutase-like Domain of DAHPS from Bacillus subtilis Complexed with Novel Inhibitor Reveals Conformational Plasticity of Active Site.

3-deoxy-D-arabino-heptulosonate-7-phosphate-synthase (DAHPS) is the first enzyme of the shikimate pathway and is responsible for the synthesis of aromatic amino acids in microorganisms. This pathway is an attractive target for antimicrobial drugs. In Bacillus subtilis, the N-terminal domain of the bifunctional DAHPS enzyme belongs to an AroQ class of chorismate mutase and is functionally homologous to the downstream AroH class chorismate mutase. This is the first structure of chorismate mutase, AroQ (BsCM_2) enzyme from Bacillus subtilis in complex with citrate and chlorogenic acid at 1.9 Å and 1.8 Å resolution, respectively. This work provides the structural basis of ligand binding into the active site of AroQ class of chorismate mutase, while accompanied by the conformational flexibility of active site loop. Molecular dynamics results showed that helix H2' undergoes uncoiling at the first turn and increases the mobility of loop L1'. The side chains of Arg45, Phe46, Arg52 and Lys76 undergo conformational changes, which may play an important role in DAHPS regulation by the formation of the domain-domain interface. Additionally, binding studies showed that the chlorogenic acid binds to BsCM_2 with a higher affinity than chorismate. These biochemical and structural findings could lead to the development of novel antimicrobial drugs.

Linear Free Energy Relationship Analysis of Transition State Mimicry by 3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) Oxime, a DAHP Synthase Inhibitor and Phosphate Mimic.

3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime replaces the ketone, is a potent inhibitor, with Ki = 1.5 µM. Linear free energy relationship (LFER) analysis of DAHP oxime inhibition using DAHP synthase mutants revealed an excellent correlation between transition state stabilization and inhibition. The equations of LFER analysis were rederived to formalize the possibility of proportional, rather than equal, changes in the free energies of transition state stabilization and inhibitor binding, in accord with the fact that the majority of LFER analyses in the literature demonstrate nonunity slopes. A slope of unity, m = 1, indicates that catalysis and inhibitor binding are equally sensitive to perturbations such as mutations or modified inhibitor/substrate structures. Slopes <1 or >1 indicate that inhibitor binding is less sensitive or more sensitive, respectively, to perturbations than is catalysis. LFER analysis using the tetramolecular specificity constant, that is, plotting log(KM,MnKM,PEPKM,E4P/kcat) versus log(Ki), revealed a slope, m, of 0.34, with r2 = 0.93. This provides evidence that DAHP oxime is mimicking the first irreversible transition state of the DAHP synthase reaction, presumably phosphate departure from the tetrahedral intermediate. This is evidence that the oxime group can act as a functional, as well as structural, mimic of phosphate groups.