The Experts below are selected from a list of 831 Experts worldwide ranked by ideXlab platform
Shelley D. Minteer - One of the best experts on this subject based on the ideXlab platform.
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tricarboxylic acid Metabolon
Methods in Enzymology, 2019Co-Authors: Shelley D. MinteerAbstract:Abstract This chapter focuses on the experimental protocols used to determine the structure of the TCA Metabolon. Since the TCA Metabolon is quite large and cannot be crystallized, X-ray crystallography and NMR-based structural biology techniques cannot be utilized. This chapter will include a discussion of the interactomics technique of cross-linking mass spectrometry used for determining the TCA Metabolon structure, including detailed procedures and the resulting Metabolon structures. It will conclude with a discussion of the lessons learned from the structure of the TCA Metabolon that are important to the field of metabolism.
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cascade kinetics of an artificial Metabolon by molecular dynamics and kinetic monte carlo
ACS Catalysis, 2018Co-Authors: Yuanchao Liu, Shelley D. Minteer, Ivana Matanovic, David P Hickey, Plamen Atanassov, Scott Calabrese BartonAbstract:Natural enzyme cascades are able to employ electrostatic channeling as an efficient mechanism for shuttling charged intermediates between sequential active sites. Application of channeling mechanisms to artificial cascades has drawn increasing attention for its potential to improve cascade design. We report a quantitative model of a two-step artificial Metabolon that accounts for molecular-level complexity. Conversion of glucose to phospho-6-gluconolactone by hexokinase and glucose-6-phosphate dehydrogenase, covalently conjugated by a cationic oligopeptide bridge, is simulated and validated by comparison to stopped-flow lag time analysis. Specifically, molecular dynamics (MD) simulations enable the calculation of energy-determined surface equilibrium constants and surface diffusivity, and a kinetic Monte Carlo (KMC) model integrated all rate constants from MD (e.g., surface diffusion and desorption rate) and experiments (e.g., turnover frequency), to estimate the product evolution on an experimental time ...
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direct evidence for Metabolon formation and substrate channeling in recombinant tca cycle enzymes
ACS Chemical Biology, 2016Co-Authors: Beyza Bulutoglu, Shelley D. Minteer, Kristen E Garcia, Scott BantaAbstract:Supramolecular assembly of enzymes into Metabolon structures is thought to enable efficient transport of reactants between active sites via substrate channeling. Recombinant versions of porcine citrate synthase (CS), mitochondrial malate dehydrogenase (mMDH), and aconitase (Aco) were found to adopt a homogeneous native-like Metabolon structure in vitro. Site-directed mutagenesis performed on highly conserved arginine residues located in the positively charged channel connecting mMDH and CS active sites led to the identification of CS(R65A) which retained high catalytic efficiency. Substrate channeling between the CS mutant and mMDH was severely impaired and the overall channeling probability decreased from 0.99 to 0.023. This work provides direct mechanistic evidence for the channeling of reaction intermediates, and disruption of this interaction would have important implications on the control of flux in central carbon metabolism.
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Krebs cycle Metabolon formation: metabolite concentration gradient enhanced compartmentation of sequential enzymes
Chemical Communications, 2015Co-Authors: Lindsey N. Pelster, Shelley D. MinteerAbstract:Dynamics of Metabolon formation in mitochondria was probed by studying diffusional motion of two sequential Krebs cycle enzymes in a microfluidic channel. Enhanced directional co-diffusion of both enzymes against a substrate concentration gradient was observed in the presence of intermediate generation. This reveals a metabolite directed compartmentation of metabolic pathways.
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Krebs cycle Metabolon: structural evidence of substrate channeling revealed by cross-linking and mass spectrometry.
Angewandte Chemie, 2014Co-Authors: Shelley D. MinteerAbstract:It has been hypothesized that the high metabolic flux in the mitochondria is due to the self-assembly of enzyme supercomplexes (called Metabolons) that channel substrates from one enzyme to another, but there has been no experimental confirmation of this structure or the channeling. A structural investigation of enzyme organization within the Krebs cycle Metabolon was accomplished by in vivo cross-linking and mass spectrometry. Eight Krebs cycle enzyme components were isolated upon chemical fixation, and interfacial residues between mitochondrial malate dehydrogenase, citrate synthase, and aconitase were identified. Using constraint protein docking, a low-resolution structure for the three-enzyme complex was achieved, as well as the two-fold symmetric octamer. Surface analysis showed formation of electrostatic channeling upon protein-protein association, which is the first structural evidence of substrate channeling in the Krebs cycle Metabolon.
Chris M Wood - One of the best experts on this subject based on the ideXlab platform.
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physiological and molecular responses of the spiny dogfish shark squalus acanthias to high environmental ammonia scavenging for nitrogen
The Journal of Experimental Biology, 2015Co-Authors: C. Michele Nawata, Patrick J Walsh, Chris M WoodAbstract:In teleosts, a branchial Metabolon links ammonia excretion to Na+ uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs (Rhag, Rhbg and Rhp2) and evaluated gill ammonia handling in Squalus acanthias. Control ammonia excretion was
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physiological and molecular responses of the spiny dogfish shark squalus acanthias to high environmental ammonia scavenging for nitrogen
The Journal of Experimental Biology, 2015Co-Authors: C. Michele Nawata, Patrick J Walsh, Chris M WoodAbstract:In teleosts, a branchial Metabolon links ammonia excretion to Na(+) uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs (Rhag, Rhbg and Rhp2) and evaluated gill ammonia handling in Squalus acanthias. Control ammonia excretion was <5% of urea-N excretion. Sharks exposed to high environmental ammonia (HEA; 1 mmol(-1) NH4HCO3) for 48 h exhibited active ammonia uptake against partial pressure and electrochemical gradients for 36 h before net excretion was re-established. Plasma total ammonia rose to seawater levels by 2 h, but dropped significantly below them by 24-48 h. Control ΔP(NH3) (the partial pressure gradient of NH3) across the gills became even more negative (outwardly directed) during HEA. Transepithelial potential increased by 30 mV, negating a parallel rise in the Nernst potential, such that the outwardly directed NH4(+) electrochemical gradient remained unchanged. Urea-N excretion was enhanced by 90% from 12 to 48 h, more than compensating for ammonia-N uptake. Expression of Rhp2 (gills, kidney) and Rhbg (kidney) did not change, but branchial Rhbg and erythrocytic Rhag declined during HEA. mRNA expression of branchial Na(+)/K(+)-ATPase (NKA) increased at 24 h and that of H(+)-ATPase decreased at 48 h, while expression of the potential Metabolon components Na(+)/H(+) exchanger2 (NHE2) and carbonic anhydrase IV (CA-IV) remained unchanged. We propose that the gill of this nitrogen-limited predator is poised not only to minimize nitrogen loss by low efflux permeability to urea and ammonia but also to scavenge ammonia-N from the environment during HEA to enhance urea-N synthesis.
Alisdair R Fernie - One of the best experts on this subject based on the ideXlab platform.
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Metabolons enzyme enzyme assemblies that mediate substrate channeling and their roles in plant metabolism
Plant communications, 2021Co-Authors: Youjun Zhang, Alisdair R FernieAbstract:Abstract Metabolons are transient multi-protein complexes of sequential enzymes that mediate substrate channeling. They differ from multi-enzyme complexes in that they are dynamic, rather than permanent, and as such have considerably lower dissociation constants. Despite the fact that a huge number of Metabolons have been suggested to exist in plants, most of these claims are erroneous as only a handful of these have been proven to channel metabolites. We believe that physical protein–protein interactions between consecutive enzymes of a pathway should rather be called enzyme–enzyme assemblies. In this review, we describe how Metabolons are generally assembled by transient interactions and held together by both structural elements and non-covalent interactions. Experimental evidence for their existence comes from protein–protein interaction studies, which indicate that the enzymes physically interact, and direct substrate channeling measurements, which indicate that they functionally interact. Unfortunately, advances in cell biology and proteomics have far outstripped those in classical enzymology and flux measurements, rendering most reports reliant purely on interactome studies. Recent developments in co-fractionation mass spectrometry will likely further exacerbate this bias. Given this, only dynamic enzyme–enzyme assemblies in which both physical and functional interactions have been demonstrated should be termed Metabolons. We discuss the level of evidence for the manifold plant pathways that have been postulated to contain Metabolons and then list examples in both primary and secondary metabolism for which strong evidence has been provided to support these claims. In doing so, we pay particular attention to experimental and mathematical approaches to study Metabolons as well as complexities that arise in attempting to follow them. Finally, we discuss perspectives for improving our understanding of these fascinating but enigmatic interactions.
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a moonlighting role for enzymes of glycolysis in the co localization of mitochondria and chloroplasts
Nature Communications, 2020Co-Authors: Youjun Zhang, Corne Swart, Alexander Graf, Lee J Sweetlove, Arun Sampathkumar, Sandra Maelin Kerber, Carsten Hille, Kumar Seerangan, Alisdair R FernieAbstract:Glycolysis is one of the primordial pathways of metabolism, playing a pivotal role in energy metabolism and biosynthesis. Glycolytic enzymes are known to form transient multi-enzyme assemblies. Here we examine the wider protein-protein interactions of plant glycolytic enzymes and reveal a moonlighting role for specific glycolytic enzymes in mediating the co-localization of mitochondria and chloroplasts. Knockout mutation of phosphoglycerate mutase or enolase resulted in a significantly reduced association of the two organelles. We provide evidence that phosphoglycerate mutase and enolase form a substrate-channelling Metabolon which is part of a larger complex of proteins including pyruvate kinase. These results alongside a range of genetic complementation experiments are discussed in the context of our current understanding of chloroplast-mitochondrial interactions within photosynthetic eukaryotes.
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resolving the Metabolon is the proof in the metabolite
EMBO Reports, 2020Co-Authors: Youjun Zhang, Alisdair R FernieAbstract:Metabolons are supra-molecular complexes of metabolic enzymes and cellular structural elements. Even though the term was coined 35 years ago, the existence of Metabolons was only recently demonstrated by a combination of metabolomics and state-of-the-art mass spectrometry.
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stable and temporary enzyme complexes and Metabolons involved in energy and redox metabolism
Antioxidants & Redox Signaling, 2020Co-Authors: Youjun Zhang, Alisdair R FernieAbstract:SIGNIFICANCE: Alongside well-characterized permanent multimeric enzymes and multi-enzyme complexes relatively unstable transient enzyme-enzyme assemblies, including Metabolons, provide an important mechanism for the regulation of energy and redox metabolism. CRITICAL ISSUES: Despite the fact that enzyme-enzyme assemblies have been proposed for many decades and experimentally analyzed for at least 40 years there are very few pathways for which unequivocal evidence for the presence of metabolite channeling, the most frequently evoked reason for their formation, has been provided. Furthermore, in contrast to the stronger, permanent interactions for which a deep understanding of the subunit interface exists the mechanism(s) underlying transient enzyme-enzyme interactions remain poorly studied. Recent advances: The widespread adoption of proteomic and cell biological approaches to characterize protein-protein interaction is defining an ever-increasing number of enzyme-enzyme assemblies as well as enzyme protein interactions that likely identify factors which stabilize such complexes. Moreover, the use of microfluidic technologies provided compelling support of a role for substrate-specific chemotaxis in complex assemblies. FUTURE DIRECTIONS: Embracing current and developing technologies should render the delineation of Metabolons from other enzyme-enzyme complexes more facile. In parallel, attempts to confirm that the findings reported in microfluidic systems are indeed representative of the cellular situation will be critical to understanding the physiological circumstances requiring and evoking dynamic changes in the levels of the various transient enzyme-enzyme assemblies of the cell.
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on the role of the tricarboxylic acid cycle in plant productivity
Journal of Integrative Plant Biology, 2018Co-Authors: Youjun Zhang, Alisdair R FernieAbstract:The tricarboxylic acid (TCA) cycle is one of the canonical energy pathways of living systems, as well as being an example of a pathway in which dynamic enzyme assemblies, or Metabolons, are well characterized. The role of the enzymes have been the subject of saturated transgenesis approaches, whereby the expression of the constituent enzymes were reduced or knocked out in order to ascertain their in vivo function. Some of the resultant plants exhibited improved photosynthesis and plant growth, under controlled greenhouse conditions. In addition, overexpression of the endogenous genes, or heterologous forms of a number of the enzymes, has been carried out in tomato fruit and the roots of a range of species, and in some instances improvement in fruit yield and postharvest properties and plant performance, under nutrient limitation, have been reported, respectively. Given a number of variants, in nature, we discuss possible synthetic approaches involving introducing these variants, or at least a subset of them, into plants. We additionally discuss the likely consequences of introducing synthetic Metabolons, wherein certain pairs of reactions are artificially permanently assembled into plants, and speculate as to future strategies to further improve plant productivity by manipulation of the core metabolic pathway.
C. Michele Nawata - One of the best experts on this subject based on the ideXlab platform.
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physiological and molecular responses of the spiny dogfish shark squalus acanthias to high environmental ammonia scavenging for nitrogen
The Journal of Experimental Biology, 2015Co-Authors: C. Michele Nawata, Patrick J Walsh, Chris M WoodAbstract:In teleosts, a branchial Metabolon links ammonia excretion to Na+ uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs (Rhag, Rhbg and Rhp2) and evaluated gill ammonia handling in Squalus acanthias. Control ammonia excretion was
-
physiological and molecular responses of the spiny dogfish shark squalus acanthias to high environmental ammonia scavenging for nitrogen
The Journal of Experimental Biology, 2015Co-Authors: C. Michele Nawata, Patrick J Walsh, Chris M WoodAbstract:In teleosts, a branchial Metabolon links ammonia excretion to Na(+) uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs (Rhag, Rhbg and Rhp2) and evaluated gill ammonia handling in Squalus acanthias. Control ammonia excretion was <5% of urea-N excretion. Sharks exposed to high environmental ammonia (HEA; 1 mmol(-1) NH4HCO3) for 48 h exhibited active ammonia uptake against partial pressure and electrochemical gradients for 36 h before net excretion was re-established. Plasma total ammonia rose to seawater levels by 2 h, but dropped significantly below them by 24-48 h. Control ΔP(NH3) (the partial pressure gradient of NH3) across the gills became even more negative (outwardly directed) during HEA. Transepithelial potential increased by 30 mV, negating a parallel rise in the Nernst potential, such that the outwardly directed NH4(+) electrochemical gradient remained unchanged. Urea-N excretion was enhanced by 90% from 12 to 48 h, more than compensating for ammonia-N uptake. Expression of Rhp2 (gills, kidney) and Rhbg (kidney) did not change, but branchial Rhbg and erythrocytic Rhag declined during HEA. mRNA expression of branchial Na(+)/K(+)-ATPase (NKA) increased at 24 h and that of H(+)-ATPase decreased at 48 h, while expression of the potential Metabolon components Na(+)/H(+) exchanger2 (NHE2) and carbonic anhydrase IV (CA-IV) remained unchanged. We propose that the gill of this nitrogen-limited predator is poised not only to minimize nitrogen loss by low efflux permeability to urea and ammonia but also to scavenge ammonia-N from the environment during HEA to enhance urea-N synthesis.
Youjun Zhang - One of the best experts on this subject based on the ideXlab platform.
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Metabolons enzyme enzyme assemblies that mediate substrate channeling and their roles in plant metabolism
Plant communications, 2021Co-Authors: Youjun Zhang, Alisdair R FernieAbstract:Abstract Metabolons are transient multi-protein complexes of sequential enzymes that mediate substrate channeling. They differ from multi-enzyme complexes in that they are dynamic, rather than permanent, and as such have considerably lower dissociation constants. Despite the fact that a huge number of Metabolons have been suggested to exist in plants, most of these claims are erroneous as only a handful of these have been proven to channel metabolites. We believe that physical protein–protein interactions between consecutive enzymes of a pathway should rather be called enzyme–enzyme assemblies. In this review, we describe how Metabolons are generally assembled by transient interactions and held together by both structural elements and non-covalent interactions. Experimental evidence for their existence comes from protein–protein interaction studies, which indicate that the enzymes physically interact, and direct substrate channeling measurements, which indicate that they functionally interact. Unfortunately, advances in cell biology and proteomics have far outstripped those in classical enzymology and flux measurements, rendering most reports reliant purely on interactome studies. Recent developments in co-fractionation mass spectrometry will likely further exacerbate this bias. Given this, only dynamic enzyme–enzyme assemblies in which both physical and functional interactions have been demonstrated should be termed Metabolons. We discuss the level of evidence for the manifold plant pathways that have been postulated to contain Metabolons and then list examples in both primary and secondary metabolism for which strong evidence has been provided to support these claims. In doing so, we pay particular attention to experimental and mathematical approaches to study Metabolons as well as complexities that arise in attempting to follow them. Finally, we discuss perspectives for improving our understanding of these fascinating but enigmatic interactions.
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a moonlighting role for enzymes of glycolysis in the co localization of mitochondria and chloroplasts
Nature Communications, 2020Co-Authors: Youjun Zhang, Corne Swart, Alexander Graf, Lee J Sweetlove, Arun Sampathkumar, Sandra Maelin Kerber, Carsten Hille, Kumar Seerangan, Alisdair R FernieAbstract:Glycolysis is one of the primordial pathways of metabolism, playing a pivotal role in energy metabolism and biosynthesis. Glycolytic enzymes are known to form transient multi-enzyme assemblies. Here we examine the wider protein-protein interactions of plant glycolytic enzymes and reveal a moonlighting role for specific glycolytic enzymes in mediating the co-localization of mitochondria and chloroplasts. Knockout mutation of phosphoglycerate mutase or enolase resulted in a significantly reduced association of the two organelles. We provide evidence that phosphoglycerate mutase and enolase form a substrate-channelling Metabolon which is part of a larger complex of proteins including pyruvate kinase. These results alongside a range of genetic complementation experiments are discussed in the context of our current understanding of chloroplast-mitochondrial interactions within photosynthetic eukaryotes.
-
resolving the Metabolon is the proof in the metabolite
EMBO Reports, 2020Co-Authors: Youjun Zhang, Alisdair R FernieAbstract:Metabolons are supra-molecular complexes of metabolic enzymes and cellular structural elements. Even though the term was coined 35 years ago, the existence of Metabolons was only recently demonstrated by a combination of metabolomics and state-of-the-art mass spectrometry.
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stable and temporary enzyme complexes and Metabolons involved in energy and redox metabolism
Antioxidants & Redox Signaling, 2020Co-Authors: Youjun Zhang, Alisdair R FernieAbstract:SIGNIFICANCE: Alongside well-characterized permanent multimeric enzymes and multi-enzyme complexes relatively unstable transient enzyme-enzyme assemblies, including Metabolons, provide an important mechanism for the regulation of energy and redox metabolism. CRITICAL ISSUES: Despite the fact that enzyme-enzyme assemblies have been proposed for many decades and experimentally analyzed for at least 40 years there are very few pathways for which unequivocal evidence for the presence of metabolite channeling, the most frequently evoked reason for their formation, has been provided. Furthermore, in contrast to the stronger, permanent interactions for which a deep understanding of the subunit interface exists the mechanism(s) underlying transient enzyme-enzyme interactions remain poorly studied. Recent advances: The widespread adoption of proteomic and cell biological approaches to characterize protein-protein interaction is defining an ever-increasing number of enzyme-enzyme assemblies as well as enzyme protein interactions that likely identify factors which stabilize such complexes. Moreover, the use of microfluidic technologies provided compelling support of a role for substrate-specific chemotaxis in complex assemblies. FUTURE DIRECTIONS: Embracing current and developing technologies should render the delineation of Metabolons from other enzyme-enzyme complexes more facile. In parallel, attempts to confirm that the findings reported in microfluidic systems are indeed representative of the cellular situation will be critical to understanding the physiological circumstances requiring and evoking dynamic changes in the levels of the various transient enzyme-enzyme assemblies of the cell.
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on the role of the tricarboxylic acid cycle in plant productivity
Journal of Integrative Plant Biology, 2018Co-Authors: Youjun Zhang, Alisdair R FernieAbstract:The tricarboxylic acid (TCA) cycle is one of the canonical energy pathways of living systems, as well as being an example of a pathway in which dynamic enzyme assemblies, or Metabolons, are well characterized. The role of the enzymes have been the subject of saturated transgenesis approaches, whereby the expression of the constituent enzymes were reduced or knocked out in order to ascertain their in vivo function. Some of the resultant plants exhibited improved photosynthesis and plant growth, under controlled greenhouse conditions. In addition, overexpression of the endogenous genes, or heterologous forms of a number of the enzymes, has been carried out in tomato fruit and the roots of a range of species, and in some instances improvement in fruit yield and postharvest properties and plant performance, under nutrient limitation, have been reported, respectively. Given a number of variants, in nature, we discuss possible synthetic approaches involving introducing these variants, or at least a subset of them, into plants. We additionally discuss the likely consequences of introducing synthetic Metabolons, wherein certain pairs of reactions are artificially permanently assembled into plants, and speculate as to future strategies to further improve plant productivity by manipulation of the core metabolic pathway.