The Experts below are selected from a list of 3795 Experts worldwide ranked by ideXlab platform
Michael F Dunn - One of the best experts on this subject based on the ideXlab platform.
-
nmr crystallography of a carbanionic intermediate in Tryptophan Synthase chemical structure tautomerization and reaction specificity
Journal of the American Chemical Society, 2016Co-Authors: Bethany G Caulkins, Michael F Dunn, Robert P Young, Ryan A Kudla, Chen Yang, Thomas J Bittbauer, Baback Bastin, E Hilario, Michael J Marsella, Leonard J MuellerAbstract:Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5′-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography—the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry—to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme Tryptophan Synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indi...
-
nmr crystallography of a carbanionic intermediate in Tryptophan Synthase chemical structure tautomerization and reaction specificity
Journal of the American Chemical Society, 2016Co-Authors: Bethany G Caulkins, Robert P Young, Ryan A Kudla, Chen Yang, Thomas J Bittbauer, Baback Bastin, Michael J Marsella, Eduardo Hilario, Li Fan, Michael F DunnAbstract:Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5'-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography-the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry-to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme Tryptophan Synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cα and positive charge at C4' of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites.
-
solution state 17 o quadrupole central transition nmr spectroscopy in the active site of Tryptophan Synthase
Angewandte Chemie, 2016Co-Authors: Robert P Young, Michael F Dunn, Bethany G Caulkins, Dan Borchardt, Daryl N Bulloch, Cynthia K Larive, Leonard J MuellerAbstract:Oxygen is an essential participant in the acid-base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution-state 17O quadrupole central transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143 kDa pyridoxal-5’-phosphate-dependent enzyme Tryptophan Synthase, reactions of the α-aminoacrylate intermediate with the nucleophiles indoline and 2-aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate-cofactor π bonding network, particularly at the adjacent alpha-carbon site.
-
solution state 17o quadrupole central transition nmr spectroscopy in the active site of Tryptophan Synthase
Angewandte Chemie, 2016Co-Authors: Robert P Young, Michael F Dunn, Bethany G Caulkins, Dan Borchardt, Daryl N Bulloch, Cynthia K Larive, Leonard J MuellerAbstract:Oxygen is an essential participant in the acid-base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution-state 17O quadrupole central transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143 kDa pyridoxal-5’-phosphate-dependent enzyme Tryptophan Synthase, reactions of the α-aminoacrylate intermediate with the nucleophiles indoline and 2-aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate-cofactor π bonding network, particularly at the adjacent alpha-carbon site.
-
nmr crystallography of enzyme active sites probing chemically detailed three dimensional structure in Tryptophan Synthase
Accounts of Chemical Research, 2013Co-Authors: Leonard J Mueller, Michael F DunnAbstract:NMR crystallography--the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry--offers unprecedented insight into three-dimensional, chemically detailed structure. Initially, researchers used NMR crystallography to refine diffraction data from organic and inorganic solids. Now we are applying this technique to explore active sites in biomolecules, where it reveals chemically rich detail concerning the interactions between enzyme site residues and the reacting substrate. Researchers cannot achieve this level of detail from X-ray, NMR,or computational methodologies in isolation. For example, typical X-ray crystal structures (1.5-2.5 A resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate but do not directly identify the protonation states. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but they rely on researcher-specified chemical details. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which scientists can develop models of the active site using computational chemistry; they can then distinguish these models by comparing calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at the highest possible resolution. In this Account, we detail our first steps in the development of NMR crystallography applied to enzyme catalysis. We begin with a brief introduction to NMR crystallography and then define the process that we have employed to probe the active site in the β-subunit of Tryptophan Synthase with unprecedented atomic-level resolution. This approach has resulted in a novel structural hypothesis for the protonation state of the quinonoid intermediate in Tryptophan Synthase and its surprising role in directing the next step in the catalysis of L-Trp formation.
Ilme Schlichting - One of the best experts on this subject based on the ideXlab platform.
-
x ray and nmr crystallography in an enzyme active site the indoline quinonoid intermediate in Tryptophan Synthase
Journal of the American Chemical Society, 2011Co-Authors: Dimitri Niks, Thomas R M Barends, Douglas W Elliott, M. Qaiser Fatmi, Chiaen A Chang, Ryan A Olsen, Tatiana Domratcheva, Friedrich W. Schwarz, Yichun Wang, Ilme SchlichtingAbstract:Chemical-level details such as protonation and hybridization state are critical for understanding enzyme mechanism and function. Even at high resolution, these details are difficult to determine by X-ray crystallography alone. The chemical shift in NMR spectroscopy, however, is an extremely sensitive probe of the chemical environment, making solid-state NMR spectroscopy and X-ray crystallography a powerful combination for defining chemically detailed three-dimensional structures. Here we adopted this combined approach to determine the chemically rich crystal structure of the indoline quinonoid intermediate in the pyridoxal-5′-phosphate-dependent enzyme Tryptophan Synthase under conditions of active catalysis. Models of the active site were developed using a synergistic approach in which the structure of this reactive substrate analogue was optimized using ab initio computational chemistry in the presence of side-chain residues fixed at their crystallographically determined coordinates. Various models of c...
-
Structure and Mechanistic Implications of a Tryptophan Synthase Quinonoid Intermediate
Chembiochem : a European journal of chemical biology, 2008Co-Authors: Thomas R M Barends, Dimitri Niks, Tatiana Domratcheva, Michael F Dunn, Victor Kulik, Lars Blumenstein, Ilme SchlichtingAbstract:Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Whereas structures of other PLP-bound reaction intermediates have been determined, a high-quality structure of a quinonoid species has not been reported. We present the crystal structure of the indoline quinonoid intermediate of Tryptophan Synthase (see figure) and discuss its implications for the enzymatic mechanism and allosteric regulation.
-
betaq114n and betat110v mutations reveal a critically important role of the substrate alpha carboxylate site in the reaction specificity of Tryptophan Synthase
Biochemistry, 2007Co-Authors: Lars Blumenstein, Dimitri Niks, Tatiana Domratcheva, Michael F Dunn, Huu Ngo, Ralf Seidel, Ilme SchlichtingAbstract:In the PLP-requiring {alpha}2{beta}2 Tryptophan Synthase complex, recognition of the substrate l-Ser at the {beta}-site includes a loop structure (residues {beta}110-115) extensively H-bonded to the substrate {alpha}-carboxylate. To investigate the relationship of this subsite to catalytic function and to the regulation of substrate channeling, two loop mutants were constructed: {beta}Thr110 {yields} Val, and {beta}Gln114 {yields} Asn. The {beta}T110V mutation greatly impairs both catalytic activity in the {beta}-reaction, and allosteric communication between the {alpha}- and {beta}-sites. The crystal structure of the {beta}T110V mutant shows that the modified l-Ser carboxylate subsite has altered protein interactions that impair {beta}-site catalysis and the communication of allosteric signals between the {alpha}- and {beta}-sites. Purified {beta}Q114N consists of two species of mutant protein, one with a reddish color ({lambda}max = 506 nm). The reddish species is unable to react with l-Ser. The second {beta}Q114N species displays significant catalytic activities; however, intermediates obtained on reaction with substrate l-Ser and substrate analogues exhibit perturbed UV/vis absorption spectra. Incubation with l-Ser results in the formation of an inactive species during the first 15 min with {lambda}max 320 nm, followed by a slower conversion over 24 h to the species with ?max = 506 nm. The 320 and 506 nmmore » species originate from conversion of the {alpha}-aminoacrylate external aldimine to the internal aldimine and {alpha}-aminoacrylate, followed by the nucleophilic attack of {alpha}-aminoacrylate on C-4' of the internal aldimine to give a covalent adduct with PLP. Subsequent treatment with sodium hydroxide releases a modified coenzyme consisting of a vinylglyoxylic acid moiety linked through C-4' to the 4-position of the pyridine ring. We conclude that the shortening of the side chain accompanying the replacement of {beta}114-Gln by Asn relaxes the steric constraints that prevent this reaction in the wild-type enzyme. This study reveals a new layer of structure-function interactions essential for reaction specificity in Tryptophan Synthase.« less
-
allosteric regulation of Tryptophan Synthase channeling the internal aldimine probed by trans 3 indole 3 acrylate binding
Biochemistry, 2007Co-Authors: Patricia Casino, Thomas R M Barends, Dimitri Niks, Ilme Schlichting, Lars Blumenstein, Peng Pan, Huu Ngo, Peter S Brzovic, Michael F DunnAbstract:Substrate channeling in the Tryptophan Synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the α-site and covalent reaction at the β-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the α-site and the external aldimine, α-aminoacrylate, and quinonoid forms of the β-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3‘-acrylate (IA), and noncleavable α-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are α-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity α-site ligands, N-(4‘-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4‘-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bin...
-
synthesis and characterization of allosteric probes of substrate channeling in the Tryptophan Synthase bienzyme complex
Biochemistry, 2007Co-Authors: Huu Ngo, Thomas R M Barends, Dimitri Niks, Victor Kulik, Lars Blumenstein, Michael Weyand, Rodney Harris, Novelle Kimmich, Patricia Casino, Ilme SchlichtingAbstract:Allosteric interactions regulate substrate channeling in Salmonella typhimurium Tryptophan Synthase. The channeling of indole between the α- and β-sites via the interconnecting 25 A tunnel is regulated by allosteric signaling arising from binding of ligand to the α-site, and covalent reaction of l-Ser at the β-site. This signaling switches the α- and β-subunits between open conformations of low activity and closed conformations of high activity. Our objective is to synthesize and characterize new classes of α-site ligands (ASLs) that mimic the binding of substrates, 3-indole-d-glycerol 3‘-phosphate (IGP) or d-glyceraldehyde 3-phosphate (G3P), for use in the investigation of α-site−β-site interactions. The new synthesized IGP analogues contain an aryl group linked to an O-phosphoethanolamine moiety through amide, sulfonamide, or thiourea groups. The G3P analogue, thiophosphoglycolohydroxamate, contains a hydroxamic acid group linked to a thiophosphate moiety. Crystal structures of the internal aldimine com...
Edith Wilson Miles - One of the best experts on this subject based on the ideXlab platform.
-
the Tryptophan Synthase α2β2 complex a model for substrate channeling allosteric communication and pyridoxal phosphate catalysis
Journal of Biological Chemistry, 2013Co-Authors: Edith Wilson MilesAbstract:I reflect on my research on pyridoxal phosphate (PLP) enzymes over fifty-five years and on how I combined research with marriage and family. My Ph.D. research with Esmond E. Snell established one aspect of PLP enzyme mechanism. My postdoctoral work first with Hans L. Kornberg and then with Alton Meister characterized the structure and function of another PLP enzyme, l-aspartate β-decarboxylase. My independent research at the National Institutes of Health (NIH) since 1966 has focused on the bacterial Tryptophan Synthase α2β2 complex. The β subunit catalyzes a number of PLP-dependent reactions. We have characterized these reactions and the allosteric effects of the α subunit. We also used chemical modification to probe enzyme structure and function. Our crystallization of the Tryptophan Synthase α2β2 complex from Salmonella typhimurium led to the determination of the three-dimensional structure with Craig Hyde and David Davies at NIH in 1988. This landmark structure was the first structure of a multienzyme complex and the first structure revealing an intramolecular tunnel. The structure has provided a basis for exploring mechanisms of catalysis, channeling, and allosteric communication in the Tryptophan Synthase α2β2 complex. The structure serves as a model for many other multiprotein complexes that are important for biological processes in prokaryotes and eukaryotes.
-
pressure and temperature jump relaxation kinetics of the conformational change in salmonella typhimurium Tryptophan Synthase l serine complex large activation compressibility and heat capacity changes demonstrate the contribution of solvation
Journal of the American Chemical Society, 2008Co-Authors: Robert S Phillips, Edith Wilson Miles, Peter Mcphie, Stephane Marchal, Cedric Georges, Yves Dupont, Reinhard LangeAbstract:Tryptophan Synthase is an α2β2 multienzyme complex that exhibits coupling of the α- and β-subunit reactions by tightly controlled allosteric interactions. A wide range of parameters can affect the allosteric interactions, including monovalent cations, pH, α-site and β-site ligands, temperature, and pressure. Rapid changes in hydrostatic pressure (P-jump) and temperature (T-jump) were used to examine the effects of pressure and temperature on the rates of the interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan Synthase-l-Ser complex. The intense fluorescence emission of the Tryptophan Synthase l-Ser external aldimine complex at 495 nm, with 420 nm excitation, provides a probe of the conformational state of Trp Synthase. P-jump measurements allowed the determination of rate constants for the reactions in the presence of Na+, Na+ with benzimidazole (BZI), and NH4+. The data require a compressibility term, βo‡, to obtain good fits, especially for the NH4+ and BZI/Na+ data. T...
-
detection of open and closed conformations of Tryptophan Synthase by 15n heteronuclear single quantum coherence nuclear magnetic resonance of bound 1 15n l Tryptophan
Journal of Biological Chemistry, 2003Co-Authors: Andrew S Osborne, Edith Wilson Miles, Quincy Teng, Robert S PhillipsAbstract:1-15N-L-Tryptophan (1-15N-L-Trp) was synthesized from 15N-aniline by a Sandmeyer reaction, followed by cyclization to isatin, reduction to indole with LiAlH4, and condensation of the 15N-indole with L-serine, catalyzed by Tryptophan Synthase. 1-15N-L-Trp was complexed with wild-type Tryptophan Synthase and beta-subunit mutants, betaK87T, betaD305A, and betaE109D, in the absence or presence of the allosteric ligands sodium chloride and disodium alpha-glycerophosphate. The enzyme complexes were observed by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance (15N-HSQC NMR) spectroscopy for the presence of 1-15N-L-Trp bound to the beta-active site. No 15N-HSQC signal was detected for 1-15N-L-Trp in 10 mm triethanolamine hydrochloride buffer at pH 8. 1-15N-L-Trp in the presence of wild-type Tryptophan Synthase in the absence or presence of 50 mm sodium chloride showed a cross peak at 10.25 ppm on the 1H axis and 129 ppm on the 15N axis as a result of reduced solvent exchange for the bound 1-15N-L-Trp, consistent with formation of a closed conformation of the active site. The addition of disodium alpha-glycerophosphate produced a signal twice as intense, suggesting that the equilibrium favors the closed conformation. 15N-HSQC NMR spectra of betaK87T and betaE109D mutant Trp Synthase with 1-15N-L-Trp showed a similar cross peak either in the presence or absence of disodium alpha-glycerophosphate, indicating the preference for a closed conformation for these mutant proteins. In contrast, the betaD305A Trp Synthase mutant only showed a 15N-HSQC signal in the presence of disodium alpha-glycerophosphate. Thus, this mutant Trp Synthase favored an open conformation in the absence of disodium alpha-glycerophosphate but was able to form a closed conformation in the presence of disodium alpha-glycerophosphate. Our results demonstrate that the 15N-HSQC NMR spectra of 1-15N-L-Trp bound to Trp Synthase can be used to determine the conformational state of mutant forms in solution rapidly. In contrast, UV-visible spectra of wild-type and mutant Trp Synthase in the presence of L-Trp with NaCl and/or disodium alpha-glycerophosphate are more difficult to interpret in terms of altered conformational equilibria.
-
allosteric communication in the Tryptophan Synthase bienzyme complex roles of the β subunit aspartate 305 arginine 141 salt bridge
Biochemistry, 2003Co-Authors: Davide Ferrari, Edith Wilson Miles, Dimitri Niks, Lihong Yang, Michael F DunnAbstract:The allosteric interactions that regulate substrate channeling and catalysis in the Tryptophan Synthase bienzyme complex from Salmonella typhimurium are triggered by covalent reactions at the β-site and binding of substrate/product to the α-site. The transmission of these allosteric signals between the α- and β-catalytic sites is modulated by an ensemble of weak bonding interactions consisting of salt bridges, hydrogen bonds, and van der Waals contacts that switch the subunits between open and closed conformations. Previous work has identified a scaffolding of salt-bridges extending between the α- and β-sites consisting of αAsp 56, βLys 167, and βAsp 305. This work investigates the involvement of yet another salt bridging interaction involving the βAsp 305−βArg 141 pair via comparison of the spectroscopic, catalytic, and allosteric properties of the βD305A and βR141A mutants with the behavior of the wild-type enzyme. These mutations were found to give bienzyme complexes with impaired allosteric communicat...
-
Tryptophan Synthase: a multienzyme complex with an intramolecular tunnel.
Chemical record (New York N.Y.), 2001Co-Authors: Edith Wilson MilesAbstract:Tryptophan Synthase is a classic enzyme that channels a metabolic intermediate, indole. The crystal structure of the Tryptophan Synthase alpha2beta2 complex from Salmonella typhimurium revealed for the first time the architecture of a multienzyme complex and the presence of an intramolecular tunnel. This remarkable hydrophobic tunnel provides a likely passageway for indole from the active site of the alpha subunit, where it is produced, to the active site of the beta subunit, where it reacts with L-serine to form L-Tryptophan in a pyridoxal phosphate-dependent reaction. Rapid kinetic studies of the wild type enzyme and of channel-impaired mutant enzymes provide strong evidence for the proposed channeling mechanism. Structures of a series of enzyme-substrate intermediates at the alpha and beta active sites are elucidating enzyme mechanisms and dynamics. These structural results are providing a fascinating picture of loops opening and closing, of domain movements, and of conformational changes in the indole tunnel. Solution studies provide further evidence for ligand-induced conformational changes that send signals between the alpha and beta subunits. The combined results show that the switching of the enzyme between open and closed conformations couples the catalytic reactions at the alpha and beta active sites and prevents the escape of indole.
Frances H. Arnold - One of the best experts on this subject based on the ideXlab platform.
-
Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation.
Journal of the American Chemical Society, 2019Co-Authors: Markus Dick, Nicholas S. Sarai, Michael W. Martynowycz, Tamir Gonen, Frances H. ArnoldAbstract:We previously engineered the β-subunit of Tryptophan Synthase (TrpB), which catalyzes the condensation of l-serine and indole to l-Tryptophan, to synthesize a range of noncanonical amino acids from...
-
tailoring Tryptophan Synthase trpb for selective quaternary carbon bond formation
ChemRxiv, 2019Co-Authors: Markus Dick, Nicholas S. Sarai, Michael W. Martynowycz, Tamir Gonen, Frances H. ArnoldAbstract:We previously engineered the Tryptophan Synthase beta-subunit (TrpB), which catalyzes the condensation reaction between L-serine and indole to form L-Tryptophan, to synthesize a range of modified Tryptophans from serine and indole derivatives. In this study, we used directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C–C bonds leading to new quaternary stereocenters. At first, the TrpBs that could use 3-substituted oxindoles preferentially formed N–C bonds by attacking the oxindole N1 atom. We found, however, that protecting the nitrogen encouraged evolution towards C-alkylation, which persisted even when this protection was removed. After seven rounds of evolution leading to a 400-fold improvement in activity, variant Pfquat efficiently alkylates 3-substituted oxindoles to selectively form new stereocenters at the γ-position of the amino acid products. The configuration of the new γ-stereocenter of one of the products was determined from the crystal structure obtained by microcrystal electron diffraction (MicroED). Substrates structurally related to 3-methyloxindole such as lactones and ketones can also be used by the enzyme for quaternary carbon bond formation, where the biocatalyst exhibits excellent regioselectivity for the tertiary carbon atom. Highly thermostable and expressed at > 500 mg/L E. coli culture, TrpB Pfquat provides an efficient and environmentally-friendly platform for the preparation of noncanonical amino acids bearing quaternary carbons.
-
Tryptophan Synthase uses an atypical mechanism to achieve substrate specificity
Biochemistry, 2016Co-Authors: Andrew R Buller, Paul Van Roye, Javier Murcianocalles, Frances H. ArnoldAbstract:Tryptophan Synthase (TrpS) catalyzes the final steps in the biosynthesis of l-Tryptophan from l-serine (Ser) and indole-3-glycerol phosphate (IGP). We report that native TrpS can also catalyze a productive reaction with l-threonine (Thr), leading to (2S,3S)-β-methylTryptophan. Surprisingly, β-substitution occurs in vitro with a 3.4-fold higher catalytic efficiency for Ser over Thr using saturating indole, despite a >82000-fold preference for Ser in direct competition using IGP. Structural data identify a novel product binding site, and kinetic experiments clarify the atypical mechanism of specificity: Thr binds efficiently but decreases the affinity for indole and disrupts the allosteric signaling that regulates the catalytic cycle.
-
a panel of trpb biocatalysts derived from Tryptophan Synthase through the transfer of mutations that mimic allosteric activation
Angewandte Chemie, 2016Co-Authors: Javier Murcianocalles, Andrew R Buller, David K Romney, Sabine Brinkmannchen, Frances H. ArnoldAbstract:Naturally occurring enzyme homologues often display highly divergent activity with non-natural substrates. Exploiting this diversity with enzymes engineered for new or altered function, however, is laborious because the engineering must be replicated for each homologue. A small set of mutations of the Tryptophan Synthase β-subunit (TrpB) from Pyrococcus furiosus, which mimics the activation afforded by binding of the α-subunit, was demonstrated to have a similar activating effect in different TrpB homologues with as little as 57 % sequence identity. Kinetic and spectroscopic analyses indicate that the mutations function through the same mechanism: mimicry of α-subunit binding. From these enzymes, we identified a new TrpB catalyst that displays a remarkably broad activity profile in the synthesis of 5-substituted Tryptophans. This demonstrates that allosteric activation can be recapitulated throughout a protein family to explore natural sequence diversity for desirable biocatalytic transformations.
-
synthesis of β branched Tryptophan analogues using an engineered subunit of Tryptophan Synthase
Journal of the American Chemical Society, 2016Co-Authors: Michael Herger, Andrew R Buller, Paul Van Roye, David K Romney, Sabine Brinkmannchen, Frances H. ArnoldAbstract:We report that l-threonine may substitute for l-serine in the β-substitution reaction of an engineered subunit of Tryptophan Synthase from Pyrococcus furiosus, yielding (2S,3S)-β-methylTryptophan (β-MeTrp) in a single step. The trace activity of the wild-type β-subunit on this substrate was enhanced more than 1000-fold by directed evolution. Structural and spectroscopic data indicate that this increase is correlated with stabilization of the electrophilic aminoacrylate intermediate. The engineered biocatalyst also reacts with a variety of indole analogues and thiophenol for diastereoselective C–C, C–N, and C–S bond-forming reactions. This new activity circumvents the 3-enzyme pathway that produces β-MeTrp in nature and offers a simple and expandable route to preparing derivatives of this valuable building block.
Thilo Stehle - One of the best experts on this subject based on the ideXlab platform.
-
site directed mutagenesis switching a dimethylallyl Tryptophan Synthase to a specific tyrosine c3 prenylating enzyme
Journal of Biological Chemistry, 2015Co-Authors: Aili Fan, Edyta Stec, Georg Zocher, Thilo StehleAbstract:Abstract The Tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl Tryptophan Synthase) from Aspergillus fumigatus catalyze C4- and C7-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C3-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-Tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-Tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis.
-
site directed mutagenesis switching a dimethylallyl Tryptophan Synthase to a specific tyrosine c3 prenylating enzyme
Journal of Biological Chemistry, 2015Co-Authors: Aili Fan, Edyta Stec, Georg Zocher, Thilo StehleAbstract:The Tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl Tryptophan Synthase) from Aspergillus fumigatus catalyze C4- and C7-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C3-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-Tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-Tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis.Dimethylallyl Tryptophan Synthase FgaPT2 catalyzes in nature the C4-prenylation of indole ring. Results FgaPT2 also catalyzes in vitro a regular C3-prenylation of l-tyrosine; its mutant FgaPT2_K174F showed a much higher catalytic activity toward l-tyrosine than l-Tryptophan. Conclusion Single mutation on the key amino acid switches the Tryptophan C4-prenyltransferase to a tyrosine C3-prenylating enzyme. Significance The first l-tyrosine C3-prenylating enzyme was created by molecular modeling-guided mutagenesis.
-
the structure of dimethylallyl Tryptophan Synthase reveals a common architecture of aromatic prenyltransferases in fungi and bacteria
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Ute Metzger, Edyta Stec, Georg Zocher, Christoph Schall, Inge A Unsold, Lutz Heide, Thilo StehleAbstract:Ergot alkaloids are toxins and important pharmaceuticals that are produced biotechnologically on an industrial scale. The first committed step of ergot alkaloid biosynthesis is catalyzed by dimethylallyl Tryptophan Synthase (DMATS; EC 2.5.1.34). Orthologs of DMATS are found in many fungal genomes. We report here the x-ray structure of DMATS, determined at a resolution of 1.76 Å. A complex of DMATS from Aspergillus fumigatus with its aromatic substrate L-Tryptophan and with an analogue of its isoprenoid substrate dimethylallyl diphosphate reveals the structural basis of this enzyme-catalyzed Friedel-Crafts reaction, which shows strict regiospecificity for position 4 of the indole nucleus of Tryptophan as well as unusual independence of the presence of Mg2+ ions. The 3D structure of DMATS belongs to a rare β/α barrel fold, called prenyltransferase barrel, that was recently discovered in a small group of bacterial enzymes with no sequence similarity to DMATS. These bacterial enzymes catalyze the prenylation of aromatic substrates in the biosynthesis of secondary metabolites (i.e., a reaction similar to that of DMATS).
-
the structure of dimethylallyl Tryptophan Synthase reveals a common architecture of aromatic prenyltransferases in fungi and bacteria
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Ute Metzger, Edyta Stec, Georg Zocher, Christoph Schall, Inge A Unsold, Lutz Heide, Thilo StehleAbstract:Ergot alkaloids are toxins and important pharmaceuticals that are produced biotechnologically on an industrial scale. The first committed step of ergot alkaloid biosynthesis is catalyzed by dimethylallyl Tryptophan Synthase (DMATS; EC 2.5.1.34). Orthologs of DMATS are found in many fungal genomes. We report here the x-ray structure of DMATS, determined at a resolution of 1.76 A. A complex of DMATS from Aspergillus fumigatus with its aromatic substrate L-Tryptophan and with an analogue of its isoprenoid substrate dimethylallyl diphosphate reveals the structural basis of this enzyme-catalyzed Friedel-Crafts reaction, which shows strict regiospecificity for position 4 of the indole nucleus of Tryptophan as well as unusual independence of the presence of Mg(2+) ions. The 3D structure of DMATS belongs to a rare beta/alpha barrel fold, called prenyltransferase barrel, that was recently discovered in a small group of bacterial enzymes with no sequence similarity to DMATS. These bacterial enzymes catalyze the prenylation of aromatic substrates in the biosynthesis of secondary metabolites (i.e., a reaction similar to that of DMATS).
