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Reuben J Peters - One of the best experts on this subject based on the ideXlab platform.
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magnesium specific ring expansion contraction catalysed by the class ii diterpene cyclase from pleuromutilin biosynthesis
Organic and Biomolecular Chemistry, 2020Co-Authors: Cody Lemke, Owen Whitham, Reuben J PetersAbstract:The class II diterpene cyclase (DTC) from pleuromutilin biosynthesis uniquely mediates 'A' ring contraction of the initially formed decalin bicycle, yielding mutildienyl diphosphate (MPP). Catalysis requires a Divalent Metal Cation co-factor. Intriguingly, selectively with magnesium, this DTC catalyzes ring expansion/contraction between MPP and halimadienyl diphosphate, providing some catalytic insight.
Adrian Goldman - One of the best experts on this subject based on the ideXlab platform.
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A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: AppliCation to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters
PLoS ONE, 2015Co-Authors: Cheng Ma, Zhenyu Hao, Amelia Lesiuk, Per Bullough, Simon E. Phillips, G Huysmans, Mark Bartlam, James D. Young, Yingying Wang, Adrian GoldmanAbstract:Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purifiCation on a scale suitable for structural studies. In particular, the nature and loCation of affinity tags introduced for the purifiCation of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purifiCation. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of Divalent Metal Cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.
Victoria Guixe - One of the best experts on this subject based on the ideXlab platform.
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bifunctional adp dependent phosphofructokinase glucokinase activity in the order methanococcales biochemical characterization of the mesophilic enzyme from methanococcus maripaludis
FEBS Journal, 2014Co-Authors: Victor Castrofernandez, Felipe Bravomoraga, Alejandra Herreramorande, Victoria GuixeAbstract:In some archaea, the phosphorylation of glucose and fructose 6-phosphate (fructose 6P) is carried out by enzymes that are specific for either substrate and that use ADP as phosphoryl donor. In the hyperthermophilic archaeon Methanocaldococcus jannaschii, a bifunctional enzyme able to phosphorylate glucose and fructose 6P has been described. To determine whether the ability to phosphorylate both glucose and fructose 6P is a common feature for all enzymes of the order Methanococcales, we expressed, purified and characterized the unique homologous protein of the mesophilic archaea Methanococcus maripaludis. Assay of the enzyme activity with different sugars, Metals and nucleotides allows us to conclude that the enzyme is able to phosphorylate both fructose 6P and glucose in the presence of ADP and a Divalent Metal Cation. Kinetic characterization of the enzyme revealed complex regulation by the free Mg2+ concentration and AMP, with the latter appearing to be a key metabolite. To determine whether this enzyme could have a role in gluconeogenesis, we evaluated the reversibility of both reactions and found that glucokinase activity is reversible, whereas phosphofructokinase activity is not. To determine the important residues for glucose and fructose 6P binding, we modeled the bifunctional phosphofructokinase/glucokinase enzyme from M. maripaludis and its interactions with both sugar substrates using protein–ligand docking. Comparison of the active site of the phosphofructokinase/glucokinase enzyme from M. maripaludis with the structural models constructed for all the homology sequences present in the order Methanococcales shows that all of the ADP-dependent kinases from this order would be able to phosphorylate glucose and fructose 6P, which rules out the current annotation of these enzymes as specific phosphofructokinases. Database Model data are available in the Protein Model Data Base under accession numbers PM0079106, PM0079107, PM0079108, PM0079109, PM0079110, PM0079111, PM0079112, PM0079113, PM0079114, PM0079115 and PM0079116
Guixé Leguía, Victoria Cristina - One of the best experts on this subject based on the ideXlab platform.
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Bifunctional ADP-dependent phosphofructokinase/ glucokinase activity in the order Methanococcales – biochemical characterization of the mesophilic enzyme from Methanococcus maripaludis
FEBS, 2014Co-Authors: Castro Fernández Víctor, Bravo Moraga Felipe, Herrera Morandé Alejandra, Guixé Leguía, Victoria CristinaAbstract:Artículo de publicación ISIIn some archaea, the phosphorylation of glucose and fructose 6-phosphate (fructose 6P) is carried out by enzymes that are specific for either substrate and that use ADP as phosphoryl donor. In the hyperthermophilic archaeon Methanocaldococcus jannaschii, a bifunctional enzyme able to phosphorylate glucose and fructose 6P has been described. To determine whether the ability to phosphorylate both glucose and fructose 6P is a common feature for all enzymes of the order Methanococcales, we expressed, purified and characterized the unique homologous protein of the mesophilic archaea Methanococcus maripaludis. Assay of the enzyme activity with different sugars, Metals and nucleotides allows us to conclude that the enzyme is able to phosphorylate both fructose 6P and glucose in the presence of ADP and a Divalent Metal Cation. Kinetic characterization of the enzyme revealed complex regulation by the free Mg2+ concentration and AMP, with the latter appearing to be a key metabolite. To determine whether this enzyme could have a role in gluconeogenesis, we evaluated the reversibility of both reactions and found that glucokinase activity is reversible, whereas phosphofructokinase activity is not. To determine the important residues for glucose and fructose 6P binding, we modeled the bifunctional phosphofructokinase/ glucokinase enzyme from M. maripaludis and its interactions with both sugar substrates using protein–ligand docking. Comparison of the active site of the phosphofructokinase/glucokinase enzyme from M. maripaludis with the structural models constructed for all the homology sequences present in the order Methanococcales shows that all of the ADPdependent kinases from this order would be able to phosphorylate glucose and fructose 6P, which rules out the current annotation of these enzymes as specific phosphofructokinases.This work was supported by Fondo Nacional de Desarrollo Cientifico y Tecnologico (Fondecyt, Chile) Grant 1110137 and CONICYT scholarship 24121448
Cody Lemke - One of the best experts on this subject based on the ideXlab platform.
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magnesium specific ring expansion contraction catalysed by the class ii diterpene cyclase from pleuromutilin biosynthesis
Organic and Biomolecular Chemistry, 2020Co-Authors: Cody Lemke, Owen Whitham, Reuben J PetersAbstract:The class II diterpene cyclase (DTC) from pleuromutilin biosynthesis uniquely mediates 'A' ring contraction of the initially formed decalin bicycle, yielding mutildienyl diphosphate (MPP). Catalysis requires a Divalent Metal Cation co-factor. Intriguingly, selectively with magnesium, this DTC catalyzes ring expansion/contraction between MPP and halimadienyl diphosphate, providing some catalytic insight.