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

Chook, Yuh Min; Ke, Hengming; Lipscomb, William N.

doi:10.1073/pnas.90.18.8600

Cited by 201 publications

(238 citation statements)

References 20 publications

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“…Apart from the numerous other hits with a Z score of 2-4.5, DALI search also found similarity with chorismate mutase in B. subtilis (Z score ϭ 5; Protein Data Bank code 2CHS; r.m.s.d. of 3.8 Å for 83 C␣ atoms) (28) that is a homotrimer with the monomer having a single domain that catalyzes the conversion of chorismate to prephenate in aromatic amino acid biosynthesis. There was also some structural similarity with urocanate hydratase from Pseudomonas putida (VAST score ϭ 7.8; Protein Data Bank code 1UWK; r.m.s.d.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of Mycobacterium tuberculosis D-3-Phosphoglycerate Dehydrogenase

Dey

Grant

Sacchettini

2005

Journal of Biological Chemistry

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Phosphoglycerate dehydrogenases exist in at least three different structural motifs. The first D-3-phosphoglycerate dehydrogenase structure to be determined was from Escherichia coli and is a tetramer composed of identical subunits that contain three discernable structural domains. The crystal structure of D-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis has been determined at 2.3 Å. This enzyme represents a second structural motif of the D-3-phosphoglycerate dehydrogenase family, one that contains an extended Cterminal region. This structure is also a tetramer of identical subunits, and the extended motif of 135 amino acids exists as a fourth structural domain. This intervening domain exerts quite a surprising characteristic to the structure by introducing significant asymmetry in the tetramer. The asymmetric unit is composed of two identical subunits that exist in two different conformations characterized by rotation of ϳ180°around a hinge connecting two of the four domains. This asymmetric arrangement results in the formation of two different and distinct domain interfaces between identical domains in the asymmetric unit. As a result, the surface of the intervening domain that is exposed to solvent in one subunit is turned inward in the other subunit toward the center of the structure where it makes contact with other structural elements. Significant asymmetry is also seen at the subunit level where different conformations exist at the NAD-binding site and the putative serinebinding site in the two unique subunits. 1 catalyzes the first committed step in this conversion of D-3-phosphoglycerate to phosphohydroxypyruvate utilizing NAD ϩ as a cofactor. Subsequently, phosphohydroxypyruvate is converted to phosphoserine by phosphoserine transaminase and then to L-serine by phosphoserine phosphatase (1, 2). PGDH also belongs to a family of proteins classified as 2-hydroxy acid dehydrogenases that are generally specific for substrates with a D-configuration (3). It shares sequence homology with other members of this family such as formate dehydrogenase and glycerate dehydrogenase whose structures are also known (4, 5).PGDH appears to be ubiquitously found in all organisms. It exists in at least three different basic structural motifs that do not appear to be strictly specific for organism type (6). The PGDH of some bacteria and some lower eukaryotes, such as yeast and Neurospora are similar to the Escherichia coli enzyme. In addition to substrate and nucleotide-binding domains, they possess a homologous C-terminal domain that, in E. coli, is involved in regulation of activity through binding the effector, L-serine. Other bacteria such as Mycobacterium, Bacillus subtilis, Corynebacterium, plants such as Arabidopsis, and higher order eukaryotes, including mammals, possess a large polypeptide insertion (ϳ150 -200 amino acids) in their C-terminal segment immediately following the substrate-binding domain and before the C-terminal domain. A third motif, which lacks the C-terminal regulatory domain altog...

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Section: Resultsmentioning

confidence: 99%

Crystal Structure of Mycobacterium tuberculosis D-3-Phosphoglycerate Dehydrogenase

Dey

Grant

Sacchettini

2005

Journal of Biological Chemistry

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“…Unlabeled chorismic acid was isolated from Escherichia coli strain KA12 and purified by reverse phase chromatography. 12 Mixtures of [10][11][12] C]chorismic acid and [7][8][9][10][11][12][13][14][15][16][17][18] O,10-13 C]chorismic acid (ca. 99:1) and [10][11][12] C]chorismic acid and [1,10-13 C]chorismic acid (ca.…”

Section: General Methods and Materialsmentioning

confidence: 99%

“…NaOH (1 M,0.88 mL, 0.88 mmol) was added to a solution of methyl [7][8][9][10][11][12] C]shikimate 10 in a 1:1 mixture of THF and H 2 O (12 mL). The mixture was stirred at 23 °C for 8 h. After the removal of THF by distillation in vacuo, the aqueous mixture was lyophilized.…”

Section: [10-12 C]chorismic Acid (1a)mentioning

confidence: 99%

“…99:1 mixture of [10-12 C]chorismate (1a) and [7][8][9][10][11][12][13][14][15][16][17][18] O,10-13 C]chorismate (1b) was used to determine a primary isotope effect at the site of bond breaking, whereas ca. 99:1 mixtures of [10-12 C]chorismate (1a) and [1,10-13 C]chorismate (1c) or [9,10-13 C]chorismate (1d) were used to determine the isotope effects at the sites of bond formation.…”

Section: Labeled Chorismatesmentioning

confidence: 99%

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Isotope Effects on the Enzymatic and Nonenzymatic Reactions of Chorismate

et al. 2005

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The important biosynthetic intermediate chorismate reacts thermally by two competitive pathways, one leading to 4-hydroxybenzoate via elimination of the enolpyruvyl side chain, and the other to prephenate by a facile Claisen rearrangement. Measurements with isotopically labeled chorismate derivatives indicate that both are concerted sigmatropic processes, controlled by the orientation of the enolpyruvyl group. In the elimination reaction of [4-2 H]chorismate, roughly 60% of the label was found in pyruvate after 3 h at 60 °C. Moreover, a 1.846±0.057 2 H isotope effect for the transferred hydrogen atom and a 1.0374±0.0005 18 O isotope effect for the ether oxygen show that the transition state for this process is highly asymmetric, with hydrogen atom transfer from C4 to C9 significantly less advanced than C-O bond cleavage. In the competing Claisen rearrangement, a very large 18 O isotope effect at the bond-breaking position (1.0482±0.0005) and a smaller 13 C isotope effect at the bond-making position (1.0118±0.0004) were determined. Isotope effects of similar magnitude characterized the transformations catalyzed by evolutionarily unrelated chorismate mutases from Escherichia coli and Bacillus subtilis. The enzymatic reactions, like their solution counterpart, are thus concerted [3,3]-sigmatropic processes in which C-C bond formation lags behind C-O bond cleavage. However, as substantially larger 18 O and smaller 13 C isotope effects were observed for a mutant enzyme in which chemistry is fully rate determining, the ionic active site may favor a somewhat more polarized transition state than that seen in solution.The unimolecular rearrangement of chorismate (1) to prephenate (2) is a key step in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine (Scheme 1). Formally, this reaction can be described as a pericyclic Claisen rearrangement, a 3,3-sigmatropic process that is rarely exploited in cellular metabolism. Secondary tritium isotope effects have suggested that the uncatalyzed reaction is concerted but asynchronous, with C-O bond cleavage preceding C-C bond fomation, 1 while stereochemical studies have established a chair-like geometry for the transition state. 2 These conclusions are supported by RHF/6-31* calculations. 3Chorismate mutases accelerate the chorismate to prephenate rearrangement more than a million fold. 4 Crystallographic studies have shown that these enzymes fall into two structurally distinct classes. 5 The overwhelming majority of chorismate mutases belong to the AroQ family, whose * To whom correspondence should be addressed at cleland @enzyme.wisc.edu (W.W.C.) hilvert@org.chem.ethz.ch (D.H.) Supporting Information Available. Coordinates and computational details for calculation of the theoretical isotope effects, plus derivations of equations 2 and 3 (10 pages). This material is available free of charge via the Internet at http://pubs.acs.org. The origins of the catalytic rate enhancement achieved by chorismate mutases have been elusive. The enzymatic reactions, ...

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“…The structure and function of BsCM are well characterized. The crystal structure shows a homotrimeric pseudo-␤-barrel surrounded by ␣-helices with three solvent-accessible binding sites at the subunit interfaces [15]. As has been shown in solution the three binding sites are all equal and independent [16].…”

mentioning

confidence: 59%

Quantitative evaluation of noncovalent chorismate mutase-inhibitor binding by ESI-MS

Wendt

McCombie

Daniel

et al. 2003

J. Am. Soc. Mass Spectrom.

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Electrospray time-of-flight mass spectrometry was used to quantitatively determine the dissociation constant of chorismate mutase and a transition state analogue inhibitor. This system presents a fairly complex stoichiometry because the native protein is a homotrimer with three equal and independent substrate binding sites. We can detect the chorismate mutase trimer as well as chorismate mutase-inhibitor complexes by choosing appropriate conditions in the ESI source. To verify that the protein-inhibitor complexes are specific, titration experiments with different enzyme variants and different inhibitors were performed. A plot of the number of bound inhibitors versus added inhibitor concentration revealed saturation behavior with 3:1 (inhibitor:functional trimer) stoichiometry for the TSA. The soft ESI conditions, the relatively high protein mass of 43.5 kDa, and the low charge state (high m/z) result in broad peaks, a typical problem in analyzing noncovalent protein complexes. Due to the low molecular weight of the TSA (226 Da) the peaks of the free protein and the protein with one, two or three inhibitors bound cannot be clearly resolved. For data analysis, relative peak areas of the deconvoluted spectra of chorismate mutase-inhibitor complexes were obtained by fitting appropriate peak shapes to the signals corresponding to the free enzyme and its complexes with one, two, or three inhibitor molecules. From the relative peak areas we were able to calculate a dissociation constant that agreed well with known solution-phase data. This method may be generally useful for interpreting mass spectra of noncovalent complexes that exhibit broad peaks in the high m/z range. W ith the rapid progress in genomic sequencing and proteomics, noncovalent interactions of proteins with their binding partners are becoming a major focus of attention. Especially in drug development, where large libraries of ligands (e.g. inhibitors) are often screened, a facile method for determining relative binding strengths with low substance consumption is desirable.Soft ionization mass spectrometry, such as electrospray ionization (ESI), is able to provide useful information about noncovalent assemblies involving biological macromolecules, including their complexes with small ligands or inhibitors. Numerous studies have been published on this topic, and a number of good reviews have appeared [1][2][3]. Increasingly, the question of whether dissociation constants of noncovalent complexes can be determined by mass spectrometry is being addressed (for a review see [4]). Because mass spectrometry employs gas-phase ions, conditions must be found such that the complexes survive the desolvation and ionization processes. If successful, the mass spectrometer can be considered as a detector for solutionphase chemistry. Several methods have already been published where noncovalent solution-phase interactions have been analyzed quantitatively by mass spectrometry, including mass spectrometrically detected melting curves [5], competition studies [6 -8], and ...

show abstract

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

Cited by 201 publications

References 20 publications

Crystal Structure of Mycobacterium tuberculosis D-3-Phosphoglycerate Dehydrogenase

Crystal Structure of Mycobacterium tuberculosis D-3-Phosphoglycerate Dehydrogenase

Isotope Effects on the Enzymatic and Nonenzymatic Reactions of Chorismate

Quantitative evaluation of noncovalent chorismate mutase-inhibitor binding by ESI-MS

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