Structure of a human aspartate kinase, chorismate mutase and TyrA domain.
D. PatelJ. KopecL. ShresthaFidelma FitzpatrickD.M. PinkasA. ChaikuadSarah E. Dixon-ClarkeThomas J. McCorvieN. Burgess-BrownF. von DelftC.H. ArrowsmithA.M. EdwardsC. BountraW.W. Yue
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Chorismate mutase
Chorismate mutase (CM, EC 5.4.99.5), encoded by ARO7, catalyzes the Claisen rearrangement of chorismate to prephenate in the biosynthesis of the amino acids tyrosine and phenylalanine. The small, dimeric enzyme of the yeast Saccharomyces cerevisiae is allosterically activated by tryptophan and allosterically inhibited by tyrosine. In this work, earlier data in the literature which suggested that chorismate mutase functions in osmoregulation and vacuole biogenesis were disproven. The analysis of several strains containing aro7 point mutations or deletions did not show any other function for CM but its role in amino acid biosynthesis. By fusion to the green fluorescent protein, this protein was localized in the cytoplasm as well as in the nucleus.On the protein level, the intramolecular signal transduction from the allosteric to the active sites occuring upon effector binding was investigated in more detail. Chimeric enzymes were constructed, in which the molecular hinge loop L220s connecting the allosteric and catalytic domain in the dimer interface, were subtituted by the corresponding loops from homologous fungal enzymes. Kinetic analysis verified that this structural component is critical for protein stability and distinguishes between the activation and inhibition signal. This hinge is also involved in dimerization of the protein. Substitution of hydrophobic amino acids in and near this loop by charged residues produced a stable monomeric enzyme variant. This chorismate mutase showed reduced activity and lost allosteric regulation, but the encoding gene complemented phenylalanine and tyrosine auxotrophy of an aro7 mutant strain. These results supported the theory that yeast CM originated from a monomeric, unregulated ancestral protein similar to the Escherichia coli CM by coevolution of regulatory and stabilizing elements.In order to gain further insight into the principles of protein stabilization, the chorismate mutase from Thermus thermophilus was purified and analyzed after cloning of the structural gene aroG. This enzyme was similar to the structurally unique CM from Bacillus subtilis, but in contrast to the latter CM was inhibited by tyrosine. Computer modeling studies revealed that like in other proteins enhanced hydrophilicity on the protein surface, increased hydrophobicity of residues within the tertiary structure as well as the tightening of active site loops stabilized the protein fold.
Chorismate mutase
Mutase
Shikimate pathway
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Chorismate mutase
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Chorismate mutase (EC 5.4.99.5) from the yeast Saccharomyces cerevisiae is an allosteric enzyme which can be locked in its active R (relaxed) state by a single threonine-to-isoleucine exchange at position 226. Seven new replacements of residue 226 reveal that this position is able to direct the enzyme's allosteric equilibrium, without interfering with the catalytic constant or the affinity for the activator.
Chorismate mutase
Residue (chemistry)
Mutase
Allosteric enzyme
Isoleucine
Alanine
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The active site of the allosteric chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5) from yeast Saccharomyces cerevisiae (YCM) was located by comparison with the mutase domain (ECM) of chorismate mutase/prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] (the P protein) from Escherichia coli. Active site domains of these two enzymes show very similar four-helix bundles, each of 94 residues which superimpose with a rms deviation of 1.06 A. Of the seven active site residues, four are conserved: the two arginines, which bind to the inhibitor's two carboxylates; the lysine, which binds to the ether oxygen; and the glutamate, which binds to the inhibitor's hydroxyl group in ECM and presumably in YCM. The other three residues in YCM (ECM) are Thr-242 (Ser-84), Asn-194 (Asp-48), and Glu-246 (Gln-88). This Glu-246, modeled close to the ether oxygen of chorismate in YCM, may function as a polarizing or ionizable group, which provides another facet to the catalytic mechanism.
Chorismate mutase
Phosphoglycerate mutase
Mutase
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Chorismate mutase
Docking (animal)
Transition state analog
Mutase
Rational design
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3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase-an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis-and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn(2+) and Mn(2+) + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.
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Shikimate pathway
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Electrostatic interactions play important roles in the catalysis of chorismate to prephenate by chorismate mutase. Mutation of Gln88 to glutamate in the monofunctional chorismate mutase from Escherichia coli results in an enzyme with a pH profile of activity significantly different from that of the wild type protein. To investigate whether the mutation alters the substrate binding process or the catalysis, we have directly determined the thermodynamic parameters of a transition state analogue inhibitor binding to the wild-type chorismate mutase and its Q88E mutant using isothermal titration calorimetry. The results demonstrate that solvent reorganization and hydrophobic interactions contribute the predominant free energy to inhibitor binding. The charge state of Glu88 in the Q88E mutant was experimentally determined and was shown to be protonated at pH 4.5 and ionized at pH 7.8, consistent with earlier hypotheses. Most surprisingly, inhibitor binding energetics do not exhibit significant pH dependency for both enzymes. Our findings indicate that the charge state of Glu88 has a small impact on inhibitor binding but plays an important role in the catalytic process.
Chorismate mutase
Isothermal Titration Calorimetry
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Chorismate mutase
Mutase
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ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTS-(−)-Dinitrobiphenic Acid: A Selective Inhibitor of Escherichiacoli Chorismate Mutase Based on Prephenate MimicryArifa Husain, Christophe C. Galopin, Sheng Zhang, Georg Pohnert, and Bruce GanemView Author Information Department of Chemistry and Chemical Biology Section of Biochemistry, Molecular and Cellular Biology, Cornell University Ithaca, New York 14853 Cite this: J. Am. Chem. Soc. 1999, 121, 11, 2647–2648Publication Date (Web):March 3, 1999Publication History Received17 December 1998Published online3 March 1999Published inissue 1 March 1999https://pubs.acs.org/doi/10.1021/ja984334ohttps://doi.org/10.1021/ja984334orapid-communicationACS PublicationsCopyright © 1999 American Chemical SocietyRequest reuse permissionsArticle Views318Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Assays,Chemical structure,Inhibition,Inhibitors,Peptides and proteins Get e-Alerts
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Abstract 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 ( Bs CM_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 Bs CM_2 with a higher affinity than chorismate. These biochemical and structural findings could lead to the development of novel antimicrobial drugs.
Chorismate mutase
Shikimate pathway
Mutase
Phosphofructokinase 2
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