Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner

Sasso, Severin; Ökvist, Mats; Roderer, Kathrin; Gamper, Marianne; Codoni, G.; Krengel, Ute; Kast, Peter

doi:10.1038/emboj.2009.165

Cited by 52 publications

(271 citation statements)

References 53 publications

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“…They highlight the importance of a cationic residueeither an arginine or a lysine-proximal to the ether oxygen of the substrate in the transition state. All known natural CMs, including the structurally unrelated AroQ enzymes from Escherichia coli (19), yeast (20), and Mycobacterium tuberculosis (41,42) share this feature, whereas poor catalysts like Arg90Cit or the catalytic antibody 1F7 (43) lack it. Rather than an exception to the classic view of enzymatic catalysis, CM appears to be an archetypical example of the Pauling paradigm.…”

Section: Discussionmentioning

confidence: 99%

Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis

Burschowsky

Eerde

Ökvist

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.pericyclic reaction | catalysis | enzyme mechanism | near attack conformation | X-ray crystal structures

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Section: Discussionmentioning

confidence: 99%

Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis

Burschowsky

Eerde

Ökvist

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The Bacillus subtilis and Porphyromonas gingivalis enzymes are fused to a catalytically active chorismate mutase enzyme, conferring allosteric control on DAH7PS activity by chorismate and prephenate (8). The single DAH7PS found in Mycobacterium tuberculosis is unusually sensitive to a combination of phenylalanine and tryptophan (9) and has been shown to interact non-covalently with chorismate mutase, both activating and providing a mechanism for control of chorismate mutase activity (10).…”

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confidence: 99%

Tyrosine Latching of a Regulatory Gate Affords Allosteric Control of Aromatic Amino Acid Biosynthesis

Cross

Dobson

Patchett

et al. 2011

Journal of Biological Chemistry

104

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The first step of the shikimate pathway for aromatic amino acid biosynthesis is catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Thermotoga maritima DAH7PS (TmaDAH7PS) is tetrameric, with monomer units comprised of a core catalytic (␤/␣) 8 barrel and an N-terminal domain. This enzyme is inhibited strongly by tyrosine and to a lesser extent by the presence of phenylalanine. A truncated mutant of TmaDAH7PS lacking the N-terminal domain was catalytically more active and completely insensitive to tyrosine and phenylalanine, consistent with a role for this domain in allosteric inhibition. The structure of this protein was determined to 2.0 Å . In contrast to the wild-type enzyme, this enzyme is dimeric. Wild-type TmaDAH7PS was co-crystallized with tyrosine, and the structure of this complex was determined to a resolution of 2.35 Å . Tyrosine was found to bind at the interface between two regulatory N-terminal domains, formed from diagonally located monomers of the tetramer, revealing a major reorganization of the regulatory domain with respect to the barrel relative to unliganded enzyme. This significant conformational rearrangement observed in the crystal structures was also clearly evident from small angle X-ray scattering measurements recorded in the presence and absence of tyrosine. The closed conformation adopted by the protein on tyrosine binding impedes substrate entry into the neighboring barrel, revealing an unusual tyrosine-controlled gating mechanism for allosteric control of this enzyme.3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS, 2 E.C. 2.5.1.54) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) (Fig. 1). This reaction is the first step in the shikimate pathway, which is used to synthesize chorismate, the precursor of the aromatic amino acids phenylalanine, tyrosine, and tryptophan, and of other important aromatic metabolites (1). The shikimate pathway is found in plants and microorganisms, and it has more recently been shown to function in apicomplexan parasites (49). As the shikimate pathway is absent in animals, the enzymes of this pathway have been identified as possible targets for the development of antimicrobial agents (2). Regulation of the catalytic activity of DAH7PS has been shown to be an important mechanism for control of cellular levels of aromatic compounds in microorganisms and plants (3).13 C NMR studies using whole cells of Escherichia coli have demonstrated that feedback inhibition of DAH7PS is the main mechanism for controlling carbon flow into the shikimate pathway (4). Different organisms employ various strategies for this feedback inhibition. E. coli, Salmonella typhimurium, and Neurospora crassa express three DAH7PS isozymes, each sensitive to a single aromatic amino acid (5, 6). In Saccharomyces cerevisiae, there are two DAH7PS isozymes; one is inhibited by phenylalanine, and the other is inhibited by tyrosine (7). Other org...

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“…Another significant distinction between MtuDAH7PS and all other DAH7P synthases characterized to date is its complex mechanism of allosteric regulation, which we have recently investigated in detail (22). Furthermore, the recent discovery that M. tuberculosis chorismate mutase is activated in complex with DAH7PS suggests a key role of DAH7PS in the regulatory network of aromatic metabolism of M. tuberculosis (26).…”

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confidence: 99%

Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

Reichau

Jiao

Walker

et al. 2011

Journal of Biological Chemistry

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Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-D-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å . Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.Tuberculosis remains a serious global health threat with over a million deaths per year. The recent emergence of multidrugresistant strains of Mycobacterium tuberculosis, the pathogen that causes the lung disease, highlights the need for rapid development of new antibacterial drugs to combat tuberculosis (1-4).3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) 4 is an important enzyme in M. tuberculosis and other pathogens. It catalyzes the first committed step in the shikimate pathway, which is responsible for the biosynthesis of aromatic amino acids and other essential aromatic metabolites in microorganisms, plants, and apicomplexan parasites (5-7). This pathway is absent in humans, and inhibitors of amino acid biosynthesis have been shown to be effective antimicrobial and herbicidal agents (8, 9). Gene disruption studies have demonstrated that M. tuberculosis is not viable if the shikimate pathway is not operational (10). These findings make DAH7PS an attractive target for drug development.DAH7PS catalyzes the aldol-like condensation of P-enolpyruvate and D-erythrose 4-phosphate (E4P) to yield 3-deoxy-Darabino-heptulusonate 7-phosphate (Fig. 1a). The reaction mechanism has been subject to extensive study, and many of the key details of the mechanism have been elucidated (11-16). The reactio...

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Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner

Cited by 52 publications

References 53 publications

Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis

Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis

Tyrosine Latching of a Regulatory Gate Affords Allosteric Control of Aromatic Amino Acid Biosynthesis

Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

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