The Experts below are selected from a list of 2238 Experts worldwide ranked by ideXlab platform
Gerhard H. Braus - One of the best experts on this subject based on the ideXlab platform.
-
Chorismate Mutase of Thermus thermophilus is a monofunctional AroH class enzyme inhibited by tyrosine
Archives of Microbiology, 2004Co-Authors: Kerstin Helmstaedt, Gabriele Heinrich, Rainer Merkl, Gerhard H. BrausAbstract:aroG , encoding the monofunctional Chorismate Mutase (TtCM) of the thermophilic gram-negative bacterium Thermus thermophilus , was cloned and its gene product characterized. TtCM was purified to homogeneity on an SDS polyacrylamide gel as a His-fusion protein with a deduced molecular mass of 15.8 kDa. The enzyme belongs to the rare group of AroH-type Chorismate Mutases which are mainly found in gram-positive bacteria of the Bacillus / Clostridia group and have recently also been described for gram-negative organisms. The native molecular mass is consistent with a pseudo-α/β barrel enzyme that is organized as a trimer. Comparison of the enzyme’s structure with that of its mesophilic counterpart from Bacillus revealed an increase in hydrophilicity on the protein’s surface, greater hydrophobicity in cavities within the protein, and greater restriction of conformational freedom, features that contribute to the thermal stability of this Chorismate Mutase. The kinetic data show Michaelis-Menten substrate saturation with a K _m of 290 μM, and a k _cat/ K _m value of 180 s^−1 mM^−1. TtCM was inhibited by tyrosine with a K _ i =34 μM, possibly in a competitive manner.
-
Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal Chorismate Mutase
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Kerstin Helmstaedt, William N. Lipscomb, Gabriele Heinrich, Gerhard H. BrausAbstract: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 of Catalytic Activity:Escherichia coli Aspartate Transcarbamoylase versus Yeast Chorismate Mutase
Microbiology and molecular biology reviews : MMBR, 2001Co-Authors: Kerstin Helmstaedt, Sven Krappmann, Gerhard H. BrausAbstract:Allosteric regulation of key metabolic enzymes is a fascinating field to study the structure-function relationship of induced conformational changes of proteins. In this review we compare the principles of allosteric transitions of the complex classical model aspartate transcarbamoylase (ATCase) from Escherichia coli, consisting of 12 polypeptides, and the less complicated Chorismate Mutase derived from baker's yeast, which functions as a homodimer. Chorismate Mutase presumably represents the minimal oligomerization state of a cooperative enzyme which still can be either activated or inhibited by different heterotropic effectors. Detailed knowledge of the number of possible quaternary states and a description of molecular triggers for conformational changes of model enzymes such as ATCase and Chorismate Mutase shed more and more light on allostery as an important regulatory mechanism of any living cell. The comparison of wild-type and engineered mutant enzymes reveals that current textbook models for regulation do not cover the entire picture needed to describe the function of these enzymes in detail.
-
HARO7 Encodes Chorismate Mutase of the Methylotrophic Yeast Hansenula polymorpha and Is Derepressed upon Methanol Utilization
Journal of bacteriology, 2000Co-Authors: Sven Krappmann, Ralph Pries, Gerd Gellissen, Mark Hiller, Gerhard H. BrausAbstract:The HARO7 gene of the methylotrophic, thermotolerant yeast Hansenula polymorpha was cloned by functional complementation. HARO7 encodes a monofunctional 280-amino-acid protein with Chorismate Mutase (EC 5.4.99.5) activity that catalyzes the conversion of Chorismate to prephenate, a key step in the biosynthesis of aromatic amino acids. The HARO7 gene product shows strong similarities to primary sequences of known eukaryotic Chorismate Mutase enzymes. After homologous overexpression and purification of the 32-kDa protein, its kinetic parameters (kcat = 319.1 s−1, nH = 1.56, [S]0.5 = 16.7 mM) as well as its allosteric regulatory properties were determined. Tryptophan acts as heterotropic positive effector; tyrosine is a negative-acting, heterotropic feedback inhibitor of enzyme activity. The influence of temperature on catalytic turnover and the thermal stability of the enzyme were determined and compared to features of the Chorismate Mutase enzyme of Saccharomyces cerevisiae. Using the Cre-loxP recombination system, we constructed mutant strains carrying a disrupted HARO7 gene that showed tyrosine auxotrophy and severe growth defects. The amount of the 0.9-kb HARO7 mRNA is independent of amino acid starvation conditions but increases twofold in the presence of methanol as the sole carbon source, implying a catabolite repression system acting on HARO7 expression.
-
The aroC gene of Aspergillus nidulans codes for a monofunctional, allosterically regulated Chorismate Mutase.
The Journal of biological chemistry, 1999Co-Authors: Sven Krappmann, Georg Schnappauf, Kerstin Helmstaedt, Thomas Gerstberger, Sabine E. Eckert, Bernd Hoffmann, Michael Hoppert, Gerhard H. BrausAbstract:Abstract The cDNA and the chromosomal locus of thearoC gene of Aspergillus nidulans were cloned and is the first representative of a filamentous fungal gene encoding Chorismate Mutase (EC 5.4.99.5), the enzyme at the first branch point of aromatic amino acid biosynthesis. The aroC gene complements the Saccharomyces cerevisiae aro7Δ as well as the A. nidulans aroC mutation. The gene consists of three exons interrupted by two short intron sequences. The expressed mRNA is 0.96 kilobases in length and aroC expression is not regulated on the transcriptional level under amino acid starvation conditions. aroC encodes a monofunctional polypeptide of 268 amino acids. Purification of this 30-kDa enzyme allowed determination of its kinetic parameters (k cat = 82 s−1, n H = 1.56, [S]0.5 = 2.3 mm), varying pH dependence of catalytic activity in different regulatory states, and an acidic pI value of 4.7. Tryptophan acts as heterotropic activator and tyrosine as negative acting, heterotropic feedback-inhibitor with aK i of 2.8 μm. Immunological data, homology modeling, as well as electron microscopy studies, indicate that this Chorismate Mutase has a dimeric structure like the S. cerevisiae enzyme. Site-directed mutagenesis of a crucial residue in loop220s (Asp233) revealed differences concerning the intramolecular signal transduction for allosteric regulation of enzymatic activity.
Donald Hilvert - One of the best experts on this subject based on the ideXlab platform.
-
An evolution-based model for designing Chorismate Mutase enzymes.
Science (New York N.Y.), 2020Co-Authors: William P. Russ, Peter Kast, Donald Hilvert, Matteo Figliuzzi, Christian Stocker, Pierre Barrat-charlaix, Michael Socolich, Rémi Monasson, Simona Cocco, Martin WeigtAbstract:The rational design of enzymes is an important goal for both fundamental and practical reasons. Here, we describe a process to learn the constraints for specifying proteins purely from evolutionary sequence data, design and build libraries of synthetic genes, and test them for activity in vivo using a quantitative complementation assay. For Chorismate Mutase, a key enzyme in the biosynthesis of aromatic amino acids, we demonstrate the design of natural-like catalytic function with substantial sequence diversity. Further optimization focuses the generative model toward function in a specific genomic context. The data show that sequence-based statistical models suffice to specify proteins and provide access to an enormous space of functional sequences. This result provides a foundation for a general process for evolution-based design of artificial proteins.
-
Evolution-based design of Chorismate Mutase enzymes
2020Co-Authors: William P. Russ, Donald Hilvert, Matteo Figliuzzi, Christian Stocker, Pierre Barrat-charlaix, Michael Socolich, Pater Kast, Rémi Monasson, Simona Cocco, Martin WeigtAbstract:The rational design of enzymes is an important goal for both fundamental and practical reasons. Here, we describe a design process in which we learn the constraints for specifying proteins purely from evolutionary sequence data, build libraries of synthetic genes, and test them for activity in vivo using a quantitative complementation assay. For Chorismate Mutase, a key enzyme in the biosynthesis of aromatic amino acids, we demonstrate the design of natural-like catalytic function with substantial sequence diversity. Further optimization focuses the generative model towards function in a specific genomic context. The data show that sequence-based statistical models suffice to specify proteins and provide access to an enormous space of synthetic functional sequences. This result provides a foundation for a general process for evolution-based design of artificial proteins.
-
Design, selection, and characterization of a split Chorismate Mutase
Protein Science, 2010Co-Authors: Manuel M Muller, Eva Csuhai, Peter Kast, Hajo Kries, Donald HilvertAbstract:Split proteins are versatile tools for detecting protein-protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable Chorismate Mutase (CM). Upon dissecting the helical-bundle CM from Methanococcus jannaschii into a short N-terminal helix and a 3-helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric Mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild-type like CM activity. Owing to the availability of a well characterized selection system, the simple 4-helix bundle topology, and the small size of the N-terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein-protein interactions.
-
Quantitative evaluation of noncovalent Chorismate Mutase-inhibitor binding by ESI-MS.
Journal of the American Society for Mass Spectrometry, 2003Co-Authors: Silke Wendt, Donald Hilvert, Gregor Mccombie, Jürg M. Daniel, Alexander Kienhöfer, Renato ZenobiAbstract: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.
-
Bacillus subtilis Chorismate Mutase is partially diffusion-controlled.
European journal of biochemistry, 1999Co-Authors: Patrizio Mattei, Peter Kast, Donald HilvertAbstract:The effect of viscosogens on the enzyme-catalyzed rearrangement of Chorismate to prephenate has been studied. The steady-state parameters kcat and kcat/Km for the monofunctional Chorismate Mutase from Bacillus subtilis (BsCM) decreased significantly with increasing concentrations of glycerol, whereas the 'sluggish' BsCM mutants C75A and C75S were insensitive to changes in microviscosity. The latter results rule out extraneous interactions of the viscosogen as an explanation for the effects observed with the wild-type enzyme. Additional control experiments show that neither viscosogen-induced shifts in the pH-dependence of the enzyme-catalyzed reaction nor small perturbations of the conformational equilibrium of Chorismate can account for the observed effects. Instead, BsCM appears to be limited by substrate binding and product release at low and high substrate concentrations, respectively. Analysis of the kinetic data indicates that diffusive transition states are between 30 and 40% rate-determining in these concentration regimes; the chemical step must contribute to the remaining kinetic barrier. The relatively low value of the 'on' rates for Chorismate and prephenate (approximately 2 x 106 m-1.s-1) probably reflects the need for a rare conformation of the enzyme, the ligand, or both for successful binding. Interestingly, the Chorismate Mutase domain of the bifunctional Chorismate Mutase-prephenate dehydratase from Escherichia coli, which has steady-state kinetic parameters comparable to those of BsCM but has a much less accessible active site, is insensitive to changes in viscosity and the reaction it catalyses is not diffusion-controlled.
Peter Kast - One of the best experts on this subject based on the ideXlab platform.
-
An evolution-based model for designing Chorismate Mutase enzymes.
Science (New York N.Y.), 2020Co-Authors: William P. Russ, Peter Kast, Donald Hilvert, Matteo Figliuzzi, Christian Stocker, Pierre Barrat-charlaix, Michael Socolich, Rémi Monasson, Simona Cocco, Martin WeigtAbstract:The rational design of enzymes is an important goal for both fundamental and practical reasons. Here, we describe a process to learn the constraints for specifying proteins purely from evolutionary sequence data, design and build libraries of synthetic genes, and test them for activity in vivo using a quantitative complementation assay. For Chorismate Mutase, a key enzyme in the biosynthesis of aromatic amino acids, we demonstrate the design of natural-like catalytic function with substantial sequence diversity. Further optimization focuses the generative model toward function in a specific genomic context. The data show that sequence-based statistical models suffice to specify proteins and provide access to an enormous space of functional sequences. This result provides a foundation for a general process for evolution-based design of artificial proteins.
-
Design, selection, and characterization of a split Chorismate Mutase
Protein Science, 2010Co-Authors: Manuel M Muller, Eva Csuhai, Peter Kast, Hajo Kries, Donald HilvertAbstract:Split proteins are versatile tools for detecting protein-protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable Chorismate Mutase (CM). Upon dissecting the helical-bundle CM from Methanococcus jannaschii into a short N-terminal helix and a 3-helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric Mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild-type like CM activity. Owing to the availability of a well characterized selection system, the simple 4-helix bundle topology, and the small size of the N-terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein-protein interactions.
-
Structure and Function of a Complex between Chorismate Mutase and Dahp Synthase: Efficiency Boost for the Junior Partner.
The EMBO journal, 2009Co-Authors: Severin Sasso, Mats Ökvist, Kathrin Roderer, Marianne Gamper, Giosiana Codoni, Ute Krengel, Peter KastAbstract:Chorismate Mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular Chorismate Mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active-site residues. However, its catalytic efficiency increases >100-fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate-pathway enzyme. The 2.35 A crystal structure of the MtCM–MtDS complex bound to a transition-state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active-site residues on binding to MtDS. The mutagenesis of the C-terminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The Chorismate-Mutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such non-covalent enzyme complexes comprising an AroQδ subclass Chorismate Mutase like MtCM are abundant in the bacterial order Actinomycetales.
-
1.6 A crystal structure of the secreted Chorismate Mutase from Mycobacterium tuberculosis: novel fold topology revealed.
Journal of molecular biology, 2006Co-Authors: Mats Ökvist, Peter Kast, Severin Sasso, Raja Dey, E. Grahn, Ute KrengelAbstract:The presence of exported Chorismate Mutases produced by certain organisms such as Mycobacterium tuberculosis has been shown to correlate with their pathogenicity. As such, these proteins comprise a new group of promising selective drug targets. Here, we report the high-resolution crystal structure of the secreted dimeric Chorismate Mutase from M. tuberculosis (*MtCM; encoded by Rv1885c), which represents the first 3D-structure of a member of this Chorismate Mutase family, termed the AroQg subclass. Structures are presented both for the unliganded enzyme and for a complex with a transition state analog. The protomer fold resembles the structurally characterized (dimeric) Escherichia coli Chorismate Mutase domain, but exhibits a new topology, with helix H4 of *MtCM carrying the catalytic site residue missing in the shortened helix H1. Furthermore, the structure of each *MtCM protomer is significantly more compact and only harbors one active site pocket, which is formed entirely by one polypeptide chain. Apart from the structural model, we present evidence as to how the substrate may enter the active site.
-
Bacillus subtilis Chorismate Mutase is partially diffusion-controlled.
European journal of biochemistry, 1999Co-Authors: Patrizio Mattei, Peter Kast, Donald HilvertAbstract:The effect of viscosogens on the enzyme-catalyzed rearrangement of Chorismate to prephenate has been studied. The steady-state parameters kcat and kcat/Km for the monofunctional Chorismate Mutase from Bacillus subtilis (BsCM) decreased significantly with increasing concentrations of glycerol, whereas the 'sluggish' BsCM mutants C75A and C75S were insensitive to changes in microviscosity. The latter results rule out extraneous interactions of the viscosogen as an explanation for the effects observed with the wild-type enzyme. Additional control experiments show that neither viscosogen-induced shifts in the pH-dependence of the enzyme-catalyzed reaction nor small perturbations of the conformational equilibrium of Chorismate can account for the observed effects. Instead, BsCM appears to be limited by substrate binding and product release at low and high substrate concentrations, respectively. Analysis of the kinetic data indicates that diffusive transition states are between 30 and 40% rate-determining in these concentration regimes; the chemical step must contribute to the remaining kinetic barrier. The relatively low value of the 'on' rates for Chorismate and prephenate (approximately 2 x 106 m-1.s-1) probably reflects the need for a rare conformation of the enzyme, the ligand, or both for successful binding. Interestingly, the Chorismate Mutase domain of the bifunctional Chorismate Mutase-prephenate dehydratase from Escherichia coli, which has steady-state kinetic parameters comparable to those of BsCM but has a much less accessible active site, is insensitive to changes in viscosity and the reaction it catalyses is not diffusion-controlled.
William N. Lipscomb - One of the best experts on this subject based on the ideXlab platform.
-
Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal Chorismate Mutase
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Kerstin Helmstaedt, William N. Lipscomb, Gabriele Heinrich, Gerhard H. BrausAbstract: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.
-
Substrate conformational transitions in the active site of Chorismate Mutase: their role in the catalytic mechanism.
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Hong Guo, William N. Lipscomb, Qiang Cui, Martin KarplusAbstract:Chorismate Mutase acts at the first branch-point of aromatic amino acid biosynthesis and catalyzes the conversion of Chorismate to prephenate. The results of molecular dynamics simulations of the substrate in solution and in the active site of Chorismate Mutase are reported. Two nonreactive conformers of Chorismate are found to be more stable than the reactive pseudodiaxial chair conformer in solution. It is shown by QM/MM molecular dynamics simulations, which take into account the motions of the enzyme, that when these inactive conformers are bound to the active site, they are rapidly converted to the reactive chair conformer. This result suggests that one contribution of the enzyme is to bind the more prevalent nonreactive conformers and transform them into the active form in a step before the chemical reaction. The motion of the reactive chair conformer in the active site calculated by using the QM/MM potential generates transient structures that are closer to the transition state than is the stable CHAIR conformer.
-
Separation of inhibition and activation of the allosteric yeast Chorismate Mutase
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Georg Schnappauf, William N. Lipscomb, Gerhard H. BrausAbstract: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.
-
A glutamate residue in the catalytic center of the yeast Chorismate Mutase restricts enzyme activity to acidic conditions
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Georg Schnappauf, William N. Lipscomb, Norbert Sträter, Gerhard H. BrausAbstract: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.
-
A glutamate residue in the catalytic center of the yeast Chorismate Mutase restricts enzyme activity to acidic conditions (Claisen rearrangementysite-directed mutagenesis)
1997Co-Authors: Georg Schnappauf, William N. Lipscomb, Gerhard H. BrausAbstract:Chorismate Mutase acts at the first branch- point 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 Mutaseyprephenate 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 neces- sity 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 ac- tivity 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.
Georg Schnappauf - One of the best experts on this subject based on the ideXlab platform.
-
The aroC gene of Aspergillus nidulans codes for a monofunctional, allosterically regulated Chorismate Mutase.
The Journal of biological chemistry, 1999Co-Authors: Sven Krappmann, Georg Schnappauf, Kerstin Helmstaedt, Thomas Gerstberger, Sabine E. Eckert, Bernd Hoffmann, Michael Hoppert, Gerhard H. BrausAbstract:Abstract The cDNA and the chromosomal locus of thearoC gene of Aspergillus nidulans were cloned and is the first representative of a filamentous fungal gene encoding Chorismate Mutase (EC 5.4.99.5), the enzyme at the first branch point of aromatic amino acid biosynthesis. The aroC gene complements the Saccharomyces cerevisiae aro7Δ as well as the A. nidulans aroC mutation. The gene consists of three exons interrupted by two short intron sequences. The expressed mRNA is 0.96 kilobases in length and aroC expression is not regulated on the transcriptional level under amino acid starvation conditions. aroC encodes a monofunctional polypeptide of 268 amino acids. Purification of this 30-kDa enzyme allowed determination of its kinetic parameters (k cat = 82 s−1, n H = 1.56, [S]0.5 = 2.3 mm), varying pH dependence of catalytic activity in different regulatory states, and an acidic pI value of 4.7. Tryptophan acts as heterotropic activator and tyrosine as negative acting, heterotropic feedback-inhibitor with aK i of 2.8 μm. Immunological data, homology modeling, as well as electron microscopy studies, indicate that this Chorismate Mutase has a dimeric structure like the S. cerevisiae enzyme. Site-directed mutagenesis of a crucial residue in loop220s (Asp233) revealed differences concerning the intramolecular signal transduction for allosteric regulation of enzymatic activity.
-
Tyrosine and Tryptophan Act through the Same Binding Site at the Dimer Interface of Yeast Chorismate Mutase
The Journal of biological chemistry, 1998Co-Authors: Georg Schnappauf, Sven Krappmann, Gerhard H. BrausAbstract:Abstract Tyrosine and tryptophan are the regulators of the dimeric yeast Chorismate Mutase. Biochemical studies reveal two binding sites per molecule for both effectors, tyrosine or tryptophan. A single binding site is built up by helix 8 and helices 4 and 5 of two different subunits. The binding sites have been analyzed in the active enzyme by site directed mutagenesis of critical codons of the coding gene, ARO7. Gly-141 and Ser-142, which both reside on helix 8, are involved in the binding of tyrosine or tryptophan presumably by interacting specifically with the amino- and carboxylate-groups of these amino acid effectors. Interaction with Thr-145 of helix 8 is required for a strong tyrosine binding to the allosteric site. Replacement of Arg-75, which connects helices 4 and 5 or of Arg-76, which is part of helix 5 by alanine residues, resulted in unregulated enzymes. These two residues are bonded to the carboxylate group and phenolic hydroxyl group of tyrosine, respectively, but do not interact with tryptophan by hydrogen bonding in the crystal structures. Phenylalanine, which has low binding affinity slightly activated the Chorismate Mutase. A T145V mutant Chorismate Mutase, however, showed increased activation by phenylalanine. Our results support a mechanism by which tyrosine contracts the allosteric site by interacting with its phenolic hydroxyl group. Tryptophan works in an inverse way by opening the allosteric site through the steric size of its side chain.
-
Separation of inhibition and activation of the allosteric yeast Chorismate Mutase
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Georg Schnappauf, William N. Lipscomb, Gerhard H. BrausAbstract: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.
-
A glutamate residue in the catalytic center of the yeast Chorismate Mutase restricts enzyme activity to acidic conditions
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Georg Schnappauf, William N. Lipscomb, Norbert Sträter, Gerhard H. BrausAbstract: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.
-
A glutamate residue in the catalytic center of the yeast Chorismate Mutase restricts enzyme activity to acidic conditions (Claisen rearrangementysite-directed mutagenesis)
1997Co-Authors: Georg Schnappauf, William N. Lipscomb, Gerhard H. BrausAbstract:Chorismate Mutase acts at the first branch- point 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 Mutaseyprephenate 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 neces- sity 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 ac- tivity 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.