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Edith Wilson Miles - One of the best experts on this subject based on the ideXlab platform.
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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.
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cryo crystallography of a true substrate indole 3 glycerol phosphate bound to a mutant αd60n tryptophan synthase α2β2 complex reveals the correct orientation of active site αglu49
Journal of Biological Chemistry, 1998Co-Authors: Sangkee Rhee, Edith Wilson Miles, David R DaviesAbstract:The reversible cleavage of indole-3-glycerol by the α-subunit of tryptophan synthase has been proposed to be catalyzed by αGlu49 and αAsp60. Although previous x-ray crystallographic structures of the tryptophan synthase α2β2 complex showed an interaction between the carboxylate of αAsp60 and the bound inhibitor indole-3-propanol phosphate, the carboxylate of αGlu49 was too distant to play its proposed role. To clarify the structural and functional roles of αGlu49, we have determined crystal structures of a mutant (αD60N) α2β2 complex in the presence and absence of the true substrate, indole-3-glycerol phosphate. The Enzyme in the crystal cleaves indole-3-glycerol phosphate very slowly at room temperature but not under cryo-conditions of 95 K. The structure of the complex with the true substrate obtained by cryo-crystallography reveals that indole-3-glycerol phosphate and indole-3-propanol phosphate have similar binding modes but different torsion angles. Most importantly, the side chain of αGlu49 interacts with 3-hydroxyl group of indole-3-glycerol phosphate as proposed. The movement of the side chain of αGlu49 into an extended conformation upon binding the true substrate provides evidence for an induced fit Mechanism. Our results demonstrate how cryo-crystallography and mutagenesis can provide insight into Enzyme Mechanism.
Nancy C Horton - One of the best experts on this subject based on the ideXlab platform.
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the run on oligomer filament Enzyme Mechanism of sgrai part 1 assembly kinetics of the run on oligomer filament
Journal of Biological Chemistry, 2018Co-Authors: Chad K Park, Jonathan L Sanchez, Claudia Barahona, Emilia L Basantes, Juan A Sanchez, Christian Hernandez, Nancy C HortonAbstract:Filament or run-on oligomer formation by metabolic Enzymes is now recognized as a widespread phenomenon having potentially unique Enzyme regulatory properties and biological roles, and its dysfunction is implicated in human diseases such as cancer, diabetes, and developmental disorders. SgrAI is a bacterial allosteric type II restriction endonuclease that binds to invading phage DNA, may protect the host DNA from off-target cleavage activity, and forms run-on oligomeric filaments with enhanced DNA-cleavage activity and altered DNA sequence specificity. However, the Mechanisms of SgrAI filament growth, cooperativity in filament formation, sequestration of Enzyme activity, and advantages over other filament Mechanisms remain unknown. In this first of a two-part series, we developed methods and models to derive association and dissociation rate constants of DNA-bound SgrAI in run-on oligomers and addressed the specific questions of cooperativity and filament growth Mechanisms. We show that the derived rate constants are consistent with the run-on oligomer sizes determined by EM analysis and are most consistent with a noncooperative growth mode of the run-on oligomer. These models and methods are extended in the accompanying article to include the full DNA-cleavage pathway and address specific questions related to the run-on oligomer Mechanism including the sequestration of DNA-cleavage activity and trapping of products.
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the run on oligomer filament Enzyme Mechanism of sgrai part 2 kinetic modeling of the full dna cleavage pathway
Journal of Biological Chemistry, 2018Co-Authors: Chad K Park, Jonathan L Sanchez, Claudia Barahona, Emilia L Basantes, Juan A Sanchez, Christian Hernandez, Nancy C HortonAbstract:Filament or run-on oligomer formation by Enzymes is now recognized as a widespread phenomenon with potentially unique Enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that forms run-on oligomeric filaments with activated DNA cleavage activity and altered DNA sequence specificity. In this two-part work, we measure individual steps in the run-on oligomer filament Mechanism to address specific questions of cooperativity, trapping, filament growth Mechanisms, and sequestration of activity using fluorophore-labeled DNA, kinetic FRET measurements, and reaction modeling with global data fitting. The final models and rate constants show that the assembly step involving association of SgrAI-DNA complexes into the run-on oligomer filament is relatively slow (3-4 orders of magnitude slower than diffusion limited) and rate-limiting at low to moderate concentrations of SgrAI-DNA. The disassembly step involving dissociation of complexes of SgrAI-DNA from each other in the run-on oligomer filament is the next slowest step but is fast enough to limit the residence time of any one copy of SgrAI or DNA within the dynamic filament. Further, the rate constant for DNA cleavage is found to be 4 orders of magnitude faster in the run-on oligomer filament than in isolated SgrAI-DNA complexes and faster than dissociation of SgrAI-DNA complexes from the run-on oligomer filament, making the reaction efficient in that each association into the filament likely leads to DNA cleavage before filament dissociation.
Michelle C Y Chang - One of the best experts on this subject based on the ideXlab platform.
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Enzyme Mechanism as a kinetic control element for designing synthetic biofuel pathways
Nature Chemical Biology, 2011Co-Authors: Brooks B Bondwatts, Robert J Bellerose, Michelle C Y ChangAbstract:As metabolic reactions are often in equilibrium, product sequestration is often used to drive engineered pathways forward. For n-butanol, however, this is not possible; instead, introducing kinetic barriers for backwards reactions significantly increases product yield.
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Enzyme Mechanism as a kinetic control element for designing synthetic biofuel pathways
Nature Chemical Biology, 2011Co-Authors: Brooks B Bondwatts, Robert J Bellerose, Michelle C Y ChangAbstract:Living systems have evolved remarkable molecular functions that can be redesigned for in vivo chemical synthesis as we gain a deeper understanding of the underlying biochemical principles for de novo construction of synthetic pathways. We have focused on developing pathways for next-generation biofuels as they require carbon to be channeled to product at quantitative yields. However, these fatty acid-inspired pathways must manage the highly reversible nature of the Enzyme components. For targets in the biodiesel range, the equilibrium can be driven to completion by physical sequestration of an insoluble product, which is a Mechanism unavailable to soluble gasoline-sized products. In this work, we report the construction of a chimeric pathway assembled from three different organisms for the high-level production of n-butanol (4,650 ± 720 mg l⁻¹) that uses an enzymatic chemical reaction Mechanism in place of a physical step as a kinetic control element to achieve high yields from glucose (28%).
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Enzyme Mechanism as a kinetic control element for designing synthetic biofuel pathways
Nature Chemical Biology, 2011Co-Authors: Brooks B Bond-watts, Robert J Bellerose, Michelle C Y ChangAbstract:As metabolic reactions are often in equilibrium, product sequestration is often used to drive engineered pathways forward. For n -butanol, however, this is not possible; instead, introducing kinetic barriers for backwards reactions significantly increases product yield. Living systems have evolved remarkable molecular functions that can be redesigned for in vivo chemical synthesis as we gain a deeper understanding of the underlying biochemical principles for de novo construction of synthetic pathways. We have focused on developing pathways for next-generation biofuels as they require carbon to be channeled to product at quantitative yields. However, these fatty acid–inspired pathways must manage the highly reversible nature of the Enzyme components. For targets in the biodiesel range, the equilibrium can be driven to completion by physical sequestration of an insoluble product, which is a Mechanism unavailable to soluble gasoline-sized products. In this work, we report the construction of a chimeric pathway assembled from three different organisms for the high-level production of n -butanol (4,650 ± 720 mg l^−1) that uses an enzymatic chemical reaction Mechanism in place of a physical step as a kinetic control element to achieve high yields from glucose (28%).
Gerard W Canters - One of the best experts on this subject based on the ideXlab platform.
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involvement of tyr108 in the Enzyme Mechanism of the small laccase from streptomyces coelicolor
Journal of the American Chemical Society, 2012Co-Authors: Ankur Gupta, Igor Nederlof, Silvia Sottini, Armand W J W Tepper, Edgar J J Groenen, Ellen A Thomassen, Gerard W CantersAbstract:The Enzyme Mechanism of the multicopper oxidase (MCO) SLAC from Streptomyces coelicolor was investigated by structural (XRD), spectroscopic (optical, EPR), and kinetics (stopped-flow) experiments on variants in which residue Tyr108 had been replaced by Phe or Ala through site-directed mutagenesis. Contrary to the more common three-domain MCOs, a tyrosine in the two-domain SLAC is found to participate in the Enzyme Mechanism by providing an electron during oxygen reduction, giving rise to the temporary appearance of a tyrosyl radical. The relatively low k(cat)/K(M) of SLAC and the involvement of Y108 in the Enzyme Mechanism may reflect an adaptation to a milieu in which there is an imbalance between the available reducing and oxidizing co-substrates. The purported evolutionary relationship between the two-domain MCOs and human ceruloplasmin appears to extend not only to the 3D structure and the mode of binding of the Cu's in the trinuclear center, as noted before, but also to the Enzyme Mechanism.
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Structure, spectroscopy, and function of tyrosinase; comparison with hemocyanin and catechol oxidase
'Wiley', 2010Co-Authors: Armand W. W. J. Tepper, Emanuela Lonardi, Bubacco Luigi, Gerard W CantersAbstract:This article deals with tyrosinase, an Enzyme that converts monophenols into diphenols and subsequently into orthoquinones with concomitant use of molecular oxygen as the source of oxygen atoms and oxidizing equivalents. The Enzyme occurs widespread in nature. Its active center consists of a dinuclear copper site. The occurrence of tyrosinases and their biological function is reviewed. Information about primary (aa sequence, genetic organization), secondary, and tertiary structures is reviewed and, where appropriate, compared with similar information about related proteins with a dinuclear Cu site (hemocyanins, catechol oxidase). Methods to produce and purify tyrosinase are reviewed. Spectroscopic studies (optical; EPR, NMR, EXAFS) are reviewed in relation to the Enzyme Mechanism, which is discussed at the end of the article
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the Enzyme Mechanism of nitrite reductase studied at single molecule level
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Sofya Kuznetsova, Gerhild Zauner, Thijs J Aartsma, H Engelkamp, Nikos S Hatzakis, Alan E Rowan, Roeland J M Nolte, P C M Christianen, Gerard W CantersAbstract:A generic method is described for the fluorescence “readout” of the activity of single redox Enzyme molecules based on Forster resonance energy transfer from a fluorescent label to the Enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to single-molecule mechanistic studies of a wide range of redox Enzymes and the precise investigation of their internal workings.
David R Davies - One of the best experts on this subject based on the ideXlab platform.
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cryo crystallography of a true substrate indole 3 glycerol phosphate bound to a mutant αd60n tryptophan synthase α2β2 complex reveals the correct orientation of active site αglu49
Journal of Biological Chemistry, 1998Co-Authors: Sangkee Rhee, Edith Wilson Miles, David R DaviesAbstract:The reversible cleavage of indole-3-glycerol by the α-subunit of tryptophan synthase has been proposed to be catalyzed by αGlu49 and αAsp60. Although previous x-ray crystallographic structures of the tryptophan synthase α2β2 complex showed an interaction between the carboxylate of αAsp60 and the bound inhibitor indole-3-propanol phosphate, the carboxylate of αGlu49 was too distant to play its proposed role. To clarify the structural and functional roles of αGlu49, we have determined crystal structures of a mutant (αD60N) α2β2 complex in the presence and absence of the true substrate, indole-3-glycerol phosphate. The Enzyme in the crystal cleaves indole-3-glycerol phosphate very slowly at room temperature but not under cryo-conditions of 95 K. The structure of the complex with the true substrate obtained by cryo-crystallography reveals that indole-3-glycerol phosphate and indole-3-propanol phosphate have similar binding modes but different torsion angles. Most importantly, the side chain of αGlu49 interacts with 3-hydroxyl group of indole-3-glycerol phosphate as proposed. The movement of the side chain of αGlu49 into an extended conformation upon binding the true substrate provides evidence for an induced fit Mechanism. Our results demonstrate how cryo-crystallography and mutagenesis can provide insight into Enzyme Mechanism.