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At least 19 recordsLinked to original sources
PubMed2005

Crystallization and preliminary X-ray crystallographic studies of Mycobacterium tuberculosis chorismate mutase.

Chorismate mutase catalyzes the first committed step in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine in bacteria, fungi and higher plants. The recent re-annotation of the Mycobacterium tuberculosis genome has revealed the presence of a duplicate set of genes coding for chorismate mutase. The mycobacterial gene Rv1885c bears <20% sequence homology to other bacterial chorismate mutases, thus serving as a potential target for the development of inhibitors specific to the pathogen. The M. tuberculosis chorismate mutase was crystallized in space group C2 and the crystals diffracted to a resolution of 2.2 A. Matthews coefficient and self-rotation function calculations revealed the presence of two monomers in the asymmetric unit.

Chorismate Mutase↗
PubMed1982

Inhibition of chorismate mutase activity of chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes.

Inhibition data (I50 values) have been obtained for inhibitors of the chorismate mutase activity of chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes. Several 1-substituted adamantane derivatives were investigated; the order of decreasing inhibitory activity with the various substituents was -PO32- much greater than -P(OCH3)O2- greater than -CO2- greater than -CH2CO2- greater than -SO2- greater than SO3-. 3-Chloroadamantane-1-acetic acid was slightly less effective than adamantane-1-acetic acid. 2-(1-Carboxy-1,4-dihydrobenzyl)acrylic acid (19), an analogue of prephenate, was an effective inhibitor. Other substances investigated, including 2,4,10-trioxaadamantane-1-acetic acid (15), 5-enolpyruvylshikimic acid (20), and 1-(carboxyethyl)-1,4-dihydrobenzoic acid (18), failed to inhibit chorismate mutase activity under the conditions investigated.

Chemical Phenomena↗
PubMed1993

13C NMR studies of the enzyme-product complex of Bacillus subtilis chorismate mutase.

The chorismate mutase reaction is a rare enzyme-catalyzed 3,3-sigmatropic rearrangement of chorismate to prephenate. Bacillus subtilis chorismate mutase was overproduced and purified from Escherichia coli XL1-Blue (pBSCM2) using a modification of the procedure of Gray et al. (Gray, J. V., Grolinelli-Pimpaneau, B., & Knowles, J. R. (1990) Biochemistry 29, 376-383); the modification leads to minimal contaminating prephenate dehydratase activity (< 0.001%). The native molecular mass of B. subtilis chorismate mutase was determined by gel filtration to be approximately 44 kDa, indicative of a homotrimer of the 14.5-kDa subunits as determined by electrospray mass spectrometry. 13C NMR was used to study the structure of [U-13C]prephenate bound at the active site of B. subtilis chorismate mutase. All the enzyme-bound 13C NMR resonances of [U-13C]prephenate were assigned, and where possible, 1JC,Cs were quantified; [1,3,5,8-13C]prephenate and [2,6,9-13C]prephenate, prepared respectively from [1,3,5,8-13C]chorismate and [2,6,9-13C]chorismate, aided the 13C NMR resonance assignments. Enzyme-bound prephenate exhibits remarkably different chemical shifts relative to free prephenate; the chemical shift changes range from -6.6 ppm for the C6 resonance to 5.6 ppm for the C5 resonance, suggesting a strong perturbation of the C5-C6 bond. 13C NMR studies of model compounds at various pH values and in various solvents suggest that the observed 13C chemical shift changes of enzyme-bound prephenate cannot be rationalized solely on the basis of changes in the pKas of the carboxylic acid groups or hydrophobic solvation at the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus subtilis↗
PubMed1994

The monofunctional chorismate mutase from Bacillus subtilis. Structure determination of chorismate mutase and its complexes with a transition state analog and prephenate, and implications for the mechanism of the enzymatic reaction.

Structures have been determined for chorismate mutase from Bacillus subtilis and of complexes of this enzyme with product and an endo-oxabicyclic transition state analog using multiple isomorphous replacement plus partial structure phase combination and non-crystallographic averaging. In addition to 522 water molecules, the model includes 1380 of the 1524 amino acid residues of the four trimers (each containing 3 x 127 amino acid residues) in the asymmetric unit. Refinement to 1.9 A resolution yields 0.194 for R and r.m.s. deviations from ideal values of 0.014 A for bond lengths and 2.92 degrees for bond angles. The trimer resembles a beta-barrel structure in which a core beta-sheet is surrounded by helices. The structures of the two complexes locate the active sites which are at the interfaces of adjacent pairs of monomers in the trimer. These structures have been refined at 2.2 A to a crystallographic R value of 0.18 and show r.m.s. deviations from ideal values of 0.013 A for bond lengths and 2.84 degrees or 3.05 degrees for bond angles, respectively. The final models have 1398 amino acid residues, nine prephenate molecules and 503 water molecules in the product complex, and 1403 amino acid residues, 12 inhibitor molecules and 530 water molecules in the transition state complex. The active sites of all three of these structures are very similar and provide a structural basis for the biochemical studies that indicate a pericyclic mechanism for conversion of chorismate to prephenate. The absence of reactive catalytic residues on the enzyme, the selective binding of the single reactive conformation of chorismate, the stabilization of the polar transition state, and the possible role of the C-terminal region in "capping" the active site are factors which relate these structures to the million-fold rate enhancement of this reaction.

Amino Acid Sequence↗
PubMed1998

Separation of inhibition and activation of the allosteric yeast chorismate mutase.

Yeast chorismate mutase (EC 5.4.99.5) shows homotropic activation by the substrate, allosteric activation by tryptophan, and allosteric inhibition by tyrosine. In this study mutants of chorismate mutase have been found that remain sensitive to one allosteric effector (tryptophan) but insensitive to the other (tyrosine). These mutations are located in the catalytic domain: loop 220s (212-226) and helix 12 (227-251). The first example starts with the Thr-266 --> Ile mutant that had previously been shown to be locked in the activated R state. The additional mutation Ile-225 --> Thr unlocks the R state and restores the activation by tryptophan but not the inhibition by tyrosine. The second example refers to a molecular trigger for the switch between the T and R state: a hydrogen-bonded system, which stabilizes only the T state, from Tyr-234 to Glu-23 to Arg-157. Various mutants of Tyr-234, especially Tyr-234 --> Phe, are unresponsive to tyrosine but are activated by tryptophan. This separation of activation from inhibition may indicate a pathway for activation that is independent of the allosteric transition and may also be consistent with an intermediate structure between T and R states.

Allosteric Regulation↗
PubMed1975

Chorismate mutase of Chlamydomonas reinhardi. Partial purification and some properties.

Chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5) was extracted from Chlamydomonas reinhardi by sonication. Fractionation of crude sonic extracts with (NH4)2SO4 and by DEAE-cellulose and Sephadex gel chromatography indicated a single peak of chorismate mutase activity with molecular weight 61 000. The Michaelis constant for 20-fold purified enzyme was 0.46 mM. Prephenate dehydrogenase (EC 1.3.1.9) and prephenate dehydratase (EC 4.2.1.40) activities were not detected in our crude or partially purified preparations of chorismate mutase. Tyrosine (1.25 mM) inhibited chorismate mutase activity by approx. 85% in crude and partially purified preparations. Phenylalanine (1.25 mM) inhibited 20%. Tryptophan (1.25 mM) by itself had no detectable effect on chorismate mutase activity but it completely reversed inhibition by tyrosine and phenylalanine. No repression of chorismate mutase was observed when the minimal growth medium was supplemented with aromatic end products.

Chlamydomonas↗
PubMed2002

Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase.

The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212-226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212-->Asp-Phe-28-->Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase.

Allosteric Regulation↗
PubMed1979

Two components of chorismate mutase in Brevibacterium flavum.

Chorismate mutase of Brevibacterium flavum, a common enzyme in phenylalanine and tyrosine biosynthesis, was separted into two different component, A and B, with molecular weights of 250,000 and 25,000, respectively, by ammonium sulfate fractionation or gel-filtration. Both components were essential for the enzymatic activity. In the presence of the reaction substrate, chorismate, the two components associated reversibly to give an active enzyme complex with a molecular weight of 320,000. Binding sites of the feedback inhibitors, phenylalanine and tyrosine, on the enzyme were localized on component A as determined by hybridization experiments with the wild-type and mutant components. Tyrosine repressed the synthesis of component B much more strongly than that of component A, while phenylalanine did not show any significant repressive effect on either component. The wild-type strain No. 2247 had four times more component A than component B. Elution patterns in gel, DEAE-cellulose or hydroxyapatite column chromatography as well as the disc-gel electrophoretic pattern of chorismate mutase component A and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase activities completely overlapped, suggesting the presence of a bifunctional protein having the two activities. In accord with this suggestion, chorismate mutase as well as DAHP synthetase was insensitive to feedback inhibition by phenylalanine and tyrosine in all the 3-fluorophenylalanine-resistant mutants tested that excreted both phenylalanine and tyrosine. All the phenylalanine and tyrosine double auxotrophs defective in chorismate mutase lacked component B but not A.

Brevibacterium↗
PubMed2005

Purified recombinant hypothetical protein coded by open reading frame Rv1885c of Mycobacterium tuberculosis exhibits a monofunctional AroQ class of periplasmic chorismate mutase activity.

Naturally occurring variants of the enzyme chorismate mutase are known to exist that exhibit diversity in enzyme structure, regulatory properties, and association with other proteins. Chorismate mutase was not annotated in the initial genome sequence of Mycobacterium tuberculosis (Mtb) because of low sequence similarity between known chorismate mutases. Recombinant protein coded by open reading frame Rv1885c of Mtb exhibited chorismate mutase activity in vitro. Biochemical and biophysical characterization of the recombinant protein suggests its resemblance to the AroQ class of chorismate mutases, prototype examples of which include the Escherichia coli and yeast chorismate mutases. We also demonstrate that unlike the corresponding proteins of E. coli, Mtb chorismate mutase does not have any associated prephenate dehydratase or dehydrogenase activity, indicating its monofunctional nature. The Rv1885c-encoded chorismate mutase showed allosteric regulation by pathway-specific as well as cross-pathway-specific ligands, as evident from proteolytic cleavage protection and enzyme assays. The predicted N-terminal signal sequence of Mtb chorismate mutase was capable of functioning as one in E. coli, suggesting that Mtb chorismate mutase belongs to the AroQ class of chorismate mutases. It was evident that Rv1885c may not be the only enzyme with chorismate mutase enzyme function within Mtb, based on our observation of the presence of chorismate mutase activity displayed by another hypothetical protein coded by open reading frame Rv0948c, a novel instance of the existence of two monofunctional chorismate mutases ever reported in any pathogenic bacterium.

Allosteric Regulation↗
PubMed1994

The crystallization and preliminary X-ray analysis of allosteric chorismate mutase.

An allosteric chorismate mutase, the Thr226-->Ile mutant, from the yeast Saccharomyces cerevisiae has been crystallized in space group P6(1)(P6(5)) using the hanging drop vapour diffusion method at room temperature. The cell dimensions are a = b = 95.8 A, c = 157.9 A, alpha = beta = 90 degrees, gamma = 120 degrees. It contains a dimer in the crystallographic asymmetric unit. The crystal diffracts to 2.2 A resolution. A native data set has been collected to 82% completeness at this resolution.

Allosteric Regulation↗
PubMed2025

Active- and Allosteric-Site Cyclic Peptide Inhibitors of Secreted M. tuberculosis Chorismate Mutase.

The secreted Chorismate mutase enzyme of Mycobacterium tuberculosis (*MtbCM) is an underexplored potential target for the development of new antitubercular agents that are increasingly needed as antibiotic resistance rises in prevalence. As an enzyme suspected to be involved in virulence and host-pathogen interactions, disruption of its function could circumvent the difficulty of treating tuberculosis-infected granulomas. Drug development, however, is limited by novel ligand discovery. Currently, *MtbCM activity is measured by using a low throughput acid/base-mediated product derivatization absorbance assay. Here, we utilized an RNA-display affinity selection approach enabled by the Random Peptides Integrated Discovery (RaPID) system to screen a vast library of macrocyclic peptides (MCP) for novel *MtbCM ligands. Peptides identified from the RaPID selection, and analogs thereof identified by analyzing the selection population dynamics, produced a new class of *MtbCM inhibiting MCPs. Among these were two noteworthy "chorismides", whose binding modes were elucidated by X-ray crystallography. Both were potent inhibitors of the CM enzyme activity. One was identified as an allosteric binding peptide revealing a novel inhibition approach, while the other is an active-site binding peptide that when conjugated to a fluorescent probe allowed for the development of a series of alternative fluorescence-based ligand-displacement assays that can be utilized for the assessment of potential *MtbCM inhibitors.

Mycobacterium tuberculosis↗
PubMed1996

Isolation of a cDNA from tomato coding for an unregulated, cytosolic chorismate mutase.

A cDNA coding for chorismate mutase was isolated from tomato by complementing a chorismate mutase-deficient Escherichia coli strain with a cDNA library. Southern blot analysis suggests the existence of a single gene of this chorismate mutase type per haploid tomato genome. The abundance of the corresponding transcripts was highest in roots, lower in stems and cotyledons, and even lower in flowers and leaves. The activity of the protein expressed in E. coli was not regulated by the three aromatic amino acids. Characteristics of the sequence and of the enzymatic activity suggest that the identified cDNA encodes a cytosolic, unregulated CM-2 type chorismate mutase.

Amino Acid Sequence↗
PubMed1997

A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions.

Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues. The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.

Chorismate Mutase↗
PubMed1993

Crystal structures of the monofunctional chorismate mutase from Bacillus subtilis and its complex with a transition state analog.

We have solved the structure of a chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5), the 1.9-A crystal structure of the monofunctional enzyme from Bacillus subtilis. The structure determination process was an unusual one, involving 12 monomers of the enzyme in the asymmetric unit. This structure was solved by the multiple isomorphous replacement method with partial structure phase combination and molecular averaging. The final model, which includes 1380 residues and 522 water molecules in an asymmetric unit, has been refined at 1.9 A and the current crystallographic R value is 0.201. The B. subtilis chorismate mutase is a homotrimer, with beta-sheets from each monomer packing to form the core of a pseudo-alpha beta-barrel with helices on the outside of the trimer. In addition, the active sites have been located by using data from a complex with an endo-oxabicyclic inhibitor that mimics the transition state of the reaction. The structure of this complex has been refined to 2.2 A with a current R value of 0.182 for a model that includes 1388 residues, 12 inhibitor molecules, and 530 water molecules in the asymmetric unit. In each trimer, three equivalent active sites are located at the interfaces of two adjacent subunits.

Amino Acid Sequence↗
PubMed1995

New insight into the catalytic mechanism of chorismate mutases from structural studies.

Chorismate mutase catalyzes the rearrangement of chorismic acid to prephenic acid, which is the first committed step in the biosynthesis of aromatic amino acids. Its catalytic mechanism has been much studied, but is poorly understood. Recent structural information on enzymes from two species, and on an antibody that catalyzes the same reaction, has shed new light on this topic.

Animals↗
PubMed1973

Chorismate mutase from Streptomyces aureofaciens: a heat-stable enzyme.

Chorismate mutase from Streptomyces aureofaciens was purified 12-fold. This enzyme preparation did not show any activity when tested for anthranilate synthetase, prephenate dehydrogenase, or prephenate dehydratase. The catalytic activity of chorismate mutase has a broad optimum between pH 7 and 8. The initial velocity data followed regular Michaelis-Menten kinetics with a K(m) of 5.3 x 10(-4) M, and the molecular weight of the enzyme was determined by sucrose gradient centrifugation to be 50,000. Heat inactivation of chorismate mutase, which occurs above temperatures of 60 C, is reversible. The enzyme activity can be restored even when chorismate mutase is treated at the temperature of a boiling-water bath for 15 min. Heat-denatured and renatured enzymes showed the same Michaelis constant and the same molecular weight as the native enzyme. l-Phenylalanine, l-tyrosine, l-tryptophan, and metabolites of the aromatic amino acid pathway were tested as potential modifiers of chorismate mutase activity. The activity of the enzyme was inhibited by none of these substances. Chorismate mutase of S. aureofaciens was not repressed in cells grown in minimal medium supplemented with l-phenylalanine, l-tyrosine, or l-tryptophan.

Cell-Free System↗
PubMed1997

Mechanisms of catalysis and allosteric regulation of yeast chorismate mutase from crystal structures.

BACKGROUND: Chorismate mutase (CM) catalyzes the Claisen rearrangement of chorismate to prephenate, notably the only known enzymatically catalyzed pericyclic reaction in primary metabolism. Structures of the enzyme in complex with an endo-oxabicyclic transition state analogue inhibitor, previously determined for Bacillus subtilis and Escherichia coli CM, provide structural insight into the enzyme mechanism. In contrast to these bacterial CMs, yeast CM is allosterically regulated in two ways: activation by tryptophan and inhibition by tyrosine. Yeast CM exists in two allosteric states, R (active) and t (inactive). RESULTS: We have determined crystal structures of wild-type yeast CM cocrystallized with tryptophan and an endo-oxabicyclic transition state analogue inhibitor, of wild-type yeast CM co-crystallized with tyrosine and the endo-oxabicyclic transition state analogue inhibitor and of the Thr226-->Ser mutant of yeast CM in complex with tryptophan. Binding of the transition state analogue inhibitor to CM keeps the enzyme in a 'super R' state, even if the inhibitory effector tyrosine is bound to the regulatory site. CONCLUSIONS: The endo-oxabicyclic inhibitor binds to yeast CM in a similar way as it does to the distantly related CM from E. coli. The inhibitor-binding mode supports a mechanism by which polar sidechains of the enzyme bind the substrate in the pseudo-diaxial conformation, which is required for catalytic turnover. A lysine and a protonated glutamate sidechain have a critical role in the stabilization of the transition state of the pericyclic reaction. The allosteric transition from T-->R state is accompanied by a 15 degrees rotation of one of the two subunits relative to the other (where 0 degrees rotation defines the T state). This rotation causes conformational changes at the dimer interface which are transmitted to the active site. An allosteric pathway is proposed to include residues Phe28, Asp24 and Glu23, which move toward the activesite cavity in the T state. In the presence of the transition-state analogue a super R state is formed, which is characterised by a 22 degrees rotation of one subunit relative to the other.

Bacillus subtilis↗