Louise E Tailford

Publications (13)60.94 Total impact

• Article: Substrate and Metal Ion Promiscuity in Mannosylglycerate Synthase

  Morten M. Nielsen, Michael D. L. Suits, Min Yang, Conor S. Barry, Carlos Martinez-Fleites, Louise E. Tailford, James E. Flint, Claire Dumon, Benjamin G. Davis, Harry J. Gilbert, Gideon J. Davies
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  ABSTRACT: The enzymatic transfer of the sugar mannose from activated sugar donors is central to the synthesis of a wide range of biologically significant polysaccharides and glycoconjugates. In addition to their importance in cellular biology, mannosyltransferases also provide model systems with which to study catalytic mechanisms of glycosyl transfer. Mannosylglycerate synthase (MGS) catalyzes the synthesis of α-mannosyl-d-glycerate using GDP-mannose as the preferred donor species, a reaction that occurs with a net retention of anomeric configuration. Past work has shown that the Rhodothermus marinus MGS, classified as a GT78 glycosyltransferase, displays a GT-A fold and performs catalysis in a metal ion-dependent manner. MGS shows very unusual metal ion dependences with Mg2+ and Ca2+ and, to a lesser extent, Mn2+, Ni2+, and Co2+, thus facilitating catalysis. Here, we probe these dependences through kinetic and calorimetric analyses of wild-type and site-directed variants of the enzyme. Mutation of residues that interact with the guanine base of GDP are correlated with a higher kcat value, whereas substitution of His-217, a key component of the metal coordination site, results in a change in metal specificity to Mn2+. Structural analyses of MGS complexes not only provide insight into metal coordination but also how lactate can function as an alternative acceptor to glycerate. These studies highlight the role of flexible loops in the active center and the subsequent coordination of the divalent metal ion as key factors in MGS catalysis and metal ion dependence. Furthermore, Tyr-220, located on a flexible loop whose conformation is likely influenced by metal binding, also plays a critical role in substrate binding.
  Journal of Biological Chemistry 04/2011; 286(17):15155-15164. · 4.77 Impact Factor
• Source

  Available from: ox.ac.uk

  Article: Substrate and metal ion promiscuity in mannosylglycerate synthase.

  Morten M Nielsen, Michael D L Suits, Min Yang, Conor S Barry, Carlos Martinez-Fleites, Louise E Tailford, James E Flint, Claire Dumon, Benjamin G Davis, Harry J Gilbert, Gideon J Davies
  [show abstract] [hide abstract]
  ABSTRACT: The enzymatic transfer of the sugar mannose from activated sugar donors is central to the synthesis of a wide range of biologically significant polysaccharides and glycoconjugates. In addition to their importance in cellular biology, mannosyltransferases also provide model systems with which to study catalytic mechanisms of glycosyl transfer. Mannosylglycerate synthase (MGS) catalyzes the synthesis of α-mannosyl-D-glycerate using GDP-mannose as the preferred donor species, a reaction that occurs with a net retention of anomeric configuration. Past work has shown that the Rhodothermus marinus MGS, classified as a GT78 glycosyltransferase, displays a GT-A fold and performs catalysis in a metal ion-dependent manner. MGS shows very unusual metal ion dependences with Mg(2+) and Ca(2+) and, to a lesser extent, Mn(2+), Ni(2+), and Co(2+), thus facilitating catalysis. Here, we probe these dependences through kinetic and calorimetric analyses of wild-type and site-directed variants of the enzyme. Mutation of residues that interact with the guanine base of GDP are correlated with a higher k(cat) value, whereas substitution of His-217, a key component of the metal coordination site, results in a change in metal specificity to Mn(2+). Structural analyses of MGS complexes not only provide insight into metal coordination but also how lactate can function as an alternative acceptor to glycerate. These studies highlight the role of flexible loops in the active center and the subsequent coordination of the divalent metal ion as key factors in MGS catalysis and metal ion dependence. Furthermore, Tyr-220, located on a flexible loop whose conformation is likely influenced by metal binding, also plays a critical role in substrate binding.
  Journal of Biological Chemistry 02/2011; 286(17):15155-64. · 4.77 Impact Factor
• Article: Understanding How Diverse β-Mannanases Recognize Heterogeneous Substrates

  Louise E. Tailford, Valerie M.-A. Ducros, James E. Flint, Shirley M. Roberts, Carl Morland, David L. Zechel, Nicola Smith, Mads E. Bjo̷rnvad, Torben V. Borchert, Keith S. Wilson, Gideon J. Davies, Harry J. Gilbert
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  ABSTRACT: The mechanism by which polysaccharide-hydrolyzing enzymes manifest specificity toward heterogeneous substrates, in which the sequence of sugars is variable, is unclear. An excellent example of such heterogeneity is provided by the plant structural polysaccharide glucomannan, which comprises a backbone of β-1,4-linked glucose and mannose units. β-Mannanases, located in glycoside hydrolase (GH) families 5 and 26, hydrolyze glucomannan by cleaving the glycosidic bond of mannosides at the −1 subsite. The mechanism by which these enzymes select for glucose or mannose at distal subsites, which is critical to defining their substrate specificity on heterogeneous polymers, is currently unclear. Here we report the biochemical properties and crystal structures of both a GH5 mannanase and a GH26 mannanase and describe the contributions to substrate specificity in these enzymes. The GH5 enzyme, BaMan5A, derived from Bacillus agaradhaerens, can accommodate glucose or mannose at both its −2 and +1 subsites, while the GH26 Bacillus subtilis mannanase, BsMan26A, displays tight specificity for mannose at its negative binding sites. The crystal structure of BaMan5A reveals that a polar residue at the −2 subsite can make productive contact with the substrate 2-OH group in either its axial (as in mannose) or its equatorial (as in glucose) configuration, while other distal subsites do not exploit the 2-OH group as a specificity determinant. Thus, BaMan5A is able to hydrolyze glucomannan in which the sequence of glucose and mannose is highly variable. The crystal structure of BsMan26A in light of previous studies on the Cellvibrio japonicus GH26 mannanases CjMan26A and CjMan26C reveals that the tighter mannose recognition at the −2 subsite is mediated by polar interactions with the axial 2-OH group of a 4C1 ground state mannoside. Mutagenesis studies showed that variants of CjMan26A, from which these polar residues had been removed, do not distinguish between Man and Glc at the −2 subsite, while one of these residues, Arg 361, confers the elevated activity displayed by the enzyme against mannooligosaccharides. The biological rationale for the variable recognition of Man- and Glc-configured sugars by β-mannanases is discussed.
  07/2009;
• Article: Understanding how diverse beta-mannanases recognize heterogeneous substrates.

  Louise E Tailford, Valerie M-A Ducros, James E Flint, Shirley M Roberts, Carl Morland, David L Zechel, Nicola Smith, Mads E Bjørnvad, Torben V Borchert, Keith S Wilson, Gideon J Davies, Harry J Gilbert
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  ABSTRACT: The mechanism by which polysaccharide-hydrolyzing enzymes manifest specificity toward heterogeneous substrates, in which the sequence of sugars is variable, is unclear. An excellent example of such heterogeneity is provided by the plant structural polysaccharide glucomannan, which comprises a backbone of beta-1,4-linked glucose and mannose units. beta-Mannanases, located in glycoside hydrolase (GH) families 5 and 26, hydrolyze glucomannan by cleaving the glycosidic bond of mannosides at the -1 subsite. The mechanism by which these enzymes select for glucose or mannose at distal subsites, which is critical to defining their substrate specificity on heterogeneous polymers, is currently unclear. Here we report the biochemical properties and crystal structures of both a GH5 mannanase and a GH26 mannanase and describe the contributions to substrate specificity in these enzymes. The GH5 enzyme, BaMan5A, derived from Bacillus agaradhaerens, can accommodate glucose or mannose at both its -2 and +1 subsites, while the GH26 Bacillus subtilis mannanase, BsMan26A, displays tight specificity for mannose at its negative binding sites. The crystal structure of BaMan5A reveals that a polar residue at the -2 subsite can make productive contact with the substrate 2-OH group in either its axial (as in mannose) or its equatorial (as in glucose) configuration, while other distal subsites do not exploit the 2-OH group as a specificity determinant. Thus, BaMan5A is able to hydrolyze glucomannan in which the sequence of glucose and mannose is highly variable. The crystal structure of BsMan26A in light of previous studies on the Cellvibrio japonicus GH26 mannanases CjMan26A and CjMan26C reveals that the tighter mannose recognition at the -2 subsite is mediated by polar interactions with the axial 2-OH group of a (4)C(1) ground state mannoside. Mutagenesis studies showed that variants of CjMan26A, from which these polar residues had been removed, do not distinguish between Man and Glc at the -2 subsite, while one of these residues, Arg 361, confers the elevated activity displayed by the enzyme against mannooligosaccharides. The biological rationale for the variable recognition of Man- and Glc-configured sugars by beta-mannanases is discussed.
  Biochemistry 06/2009; 48(29):7009-18. · 3.42 Impact Factor
• Article: Insights into plant cell wall degradation from the genome sequence of the soil bacterium Cellvibrio japonicus.

  Robert T DeBoy, Emmanuel F Mongodin, Derrick E Fouts, Louise E Tailford, Hoda Khouri, Joanne B Emerson, Yasmin Mohamoud, Kisha Watkins, Bernard Henrissat, Harry J Gilbert, Karen E Nelson
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  ABSTRACT: The plant cell wall, which consists of a highly complex array of interconnecting polysaccharides, is the most abundant source of organic carbon in the biosphere. Microorganisms that degrade the plant cell wall synthesize an extensive portfolio of hydrolytic enzymes that display highly complex molecular architectures. To unravel the intricate repertoire of plant cell wall-degrading enzymes synthesized by the saprophytic soil bacterium Cellvibrio japonicus, we sequenced and analyzed its genome, which predicts that the bacterium contains the complete repertoire of enzymes required to degrade plant cell wall and storage polysaccharides. Approximately one-third of these putative proteins (57) are predicted to contain carbohydrate binding modules derived from 13 of the 49 known families. Sequence analysis reveals approximately 130 predicted glycoside hydrolases that target the major structural and storage plant polysaccharides. In common with that of the colonic prokaryote Bacteroides thetaiotaomicron, the genome of C. japonicus is predicted to encode a large number of GH43 enzymes, suggesting that the extensive arabinose decorations appended to pectins and xylans may represent a major nutrient source, not just for intestinal bacteria but also for microorganisms that occupy terrestrial ecosystems. The results presented here predict that C. japonicus possesses an extensive range of glycoside hydrolases, lyases, and esterases. Most importantly, the genome of C. japonicus is remarkably similar to that of the gram-negative marine bacterium, Saccharophagus degradans 2-40(T). Approximately 50% of the predicted C. japonicus plant-degradative apparatus appears to be shared with S. degradans, consistent with the utilization of plant-derived complex carbohydrates as a major substrate by both organisms.
  Journal of bacteriology 09/2008; 190(15):5455-63. · 3.94 Impact Factor
• Article: Structural and biochemical evidence for a boat-like transition state in beta-mannosidases.

  Louise E Tailford, Wendy A Offen, Nicola L Smith, Claire Dumon, Carl Morland, Julie Gratien, Marie-Pierre Heck, Robert V Stick, Yves Blériot, Andrea Vasella, Harry J Gilbert, Gideon J Davies
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  ABSTRACT: Enzyme inhibition through mimicry of the transition state is a major area for the design of new therapeutic agents. Emerging evidence suggests that many retaining glycosidases that are active on alpha- or beta-mannosides harness unusual B2,5 (boat) transition states. Here we present the analysis of 25 putative beta-mannosidase inhibitors, whose Ki values range from nanomolar to millimolar, on the Bacteroides thetaiotaomicron beta-mannosidase BtMan2A. B2,5 or closely related conformations were observed for all tightly binding compounds. Subsequent linear free energy relationships that correlate log Ki with log Km/kcat for a series of active center variants highlight aryl-substituted mannoimidazoles as powerful transition state mimics in which the binding energy of the aryl group enhances both binding and the degree of transition state mimicry. Support for a B2,5 transition state during enzymatic beta-mannosidase hydrolysis should also facilitate the design and exploitation of transition state mimics for the inhibition of retaining alpha-mannosidases--an area that is emerging for anticancer therapeutics.
  Nature Chemical Biology 06/2008; 4(5):306-12. · 14.69 Impact Factor
• Article: Mannose foraging by Bacteroides thetaiotaomicron: structure and specificity of the beta-mannosidase, BtMan2A.

  Louise E Tailford, Victoria A Money, Nicola L Smith, Claire Dumon, Gideon J Davies, Harry J Gilbert
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  ABSTRACT: The human colonic bacterium Bacteroides thetaiotaomicron, which plays an important role in maintaining human health, produces an extensive array of exo-acting glycoside hydrolases (GH), including 32 family GH2 glycoside hydrolases. Although it is likely that these enzymes enable the organism to utilize dietary and host glycans as major nutrient sources, the biochemical properties of these GH2 glycoside hydrolases are currently unclear. Here we report the biochemical properties and crystal structure of the GH2 B. thetaiotaomicron enzyme BtMan2A. Kinetic analysis demonstrates that BtMan2A is a beta-mannosidase in which substrate binding energy is provided principally by the glycone binding site, whereas aglycone recognition is highly plastic. The three-dimensional structure, determined to a resolution of 1.7 A, reveals a five-domain structure that is globally similar to the Escherichia coli LacZ beta-galactosidase. The catalytic center is housed mainly within a (beta/alpha)8 barrel although the N-terminal domain also contributes to the active site topology. The nature of the substrate-binding residues is quite distinct from other GH2 enzymes of known structure, instead they are similar to other clan GH-A enzymes specific for manno-configured substrates. Mutagenesis studies, informed by the crystal structure, identified a WDW motif in the N-terminal domain that makes a significant contribution to catalytic activity. The observation that this motif is invariant in GH2 mannosidases points to a generic role for these residues in this enzyme class. The identification of GH-A clan and GH2 specific residues in the active site of BtMan2A explains why this enzyme is able to harness substrate binding at the proximal glycone binding site more efficiently than mannan-hydrolyzing glycoside hydrolases in related enzyme families. The catalytic properties of BtMan2A are consistent with the flexible nutrient acquisition displayed by the colonic bacterium.
  Journal of Biological Chemistry 05/2007; 282(15):11291-9. · 4.77 Impact Factor
• Article: Galactomannan hydrolysis and mannose metabolism in Cellvibrio mixtus.

  Maria S J Centeno, Catarina I P D Guerreiro, Fernando M V Dias, Carl Morland, Louise E Tailford, Arun Goyal, José A M Prates, Luís M A Ferreira, Rui M H Caldeira, Emmanuel F Mongodin, Karen E Nelson, Harry J Gilbert, Carlos M G A Fontes
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  ABSTRACT: Galactomannan hydrolysis results from the concerted action of microbial endo-mannanases, manosidases and alpha-galactosidases and is a mechanism of intrinsic biological importance. Here we report the identification of a gene cluster in the aerobic soil bacterium Cellvibrio mixtus encoding enzymes involved in the degradation of this polymeric substrate. The family 27 alpha-galactosidase, termed CmAga27A, preferentially hydrolyse galactose containing polysaccharides. In addition, we have characterized an enzyme with epimerase activity, which might be responsible for the conversion of mannose into glucose. The role of the identified enzymes in the hydrolysis of galactomannan by aerobic bacteria is discussed.
  FEMS Microbiology Letters 09/2006; 261(1):123-32. · 2.04 Impact Factor
• Article: Enzymatic fingerprinting of Arabidopsis pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis (PACE).

  Christopher J Barton, Louise E Tailford, Helen Welchman, Zhinong Zhang, Harry J Gilbert, Paul Dupree, Florence Goubet
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  ABSTRACT: Plant cell wall polysaccharides vary in quantity and structure between different organs and during development. However, quantitative analysis of individual polysaccharides remains challenging, and relatively little is known about any such variation in polysaccharides in organs of the model plant Arabidopsis thaliana. We have analysed plant cell wall pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis. By highly specific enzymatic digestion of a polysaccharide in a cell wall preparation, a unique fingerprint of short oligosaccharides was produced. These oligosaccharides gave quantitative and structural information on the original polysaccharide chain. We analysed enzyme-accessible polygalacturonan (PGA), linear beta(1,4) galactan and linear alpha(1,5) arabinan in several organs of Arabidopsis: roots, young leaves, old leaves, lower and upper inflorescence stems, seeds and callus. We found that this PGA constitutes a high proportion of cell wall material (CWM), up to 15% depending on the organ. In all organs, between 60 and 80% of the PGA was highly esterified in a blockwise fashion, and surprisingly, dispersely esterified PGA was hardly detected. We found enzyme-accessible linear galactan and arabinan are both present as a minor polysaccharide in all the organs. The amount of galactan ranged from ~0.04 to 0.25% of CWM, and linear arabinan constituted between 0.015 and 0.1%. Higher levels of galactan correlated with expanding tissues, supporting the hypothesis that this polysaccharide is involved in wall extension. We show by analysis of mur4 that the methods and results presented here also provide a basis for studies of pectic polysaccharides in Arabidopsis mutants.
  Planta 07/2006; 224(1):163-74. · 3.00 Impact Factor
• Article: Galactomannan hydrolysis and mannose metabolism in Cellvibrio mixtus

  Maria S.J. Centeno, Catarina I.P.D. Guerreiro, Fernando M.V. Dias, Carl Morland, Louise E. Tailford, Arun Goyal, José A.M. Prates, Luís M.A. Ferreira, Rui M.H. Caldeira, Emmanuel F. Mongodin, Karen E. Nelson, Harry J. Gilbert, Carlos M.G.A. Fontes
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  ABSTRACT: Galactomannan hydrolysis results from the concerted action of microbial endo-mannanases, manosidases and -galactosidases and is a mechanism of intrinsic biological importance. Here we report the identification of a gene cluster in the aerobic soil bacterium Cellvibrio mixtus encoding enzymes involved in the degradation of this polymeric substrate. The family 27 -galactosidase, termed CmAga27A, preferentially hydrolyse galactose containing polysaccharides. In addition, we have characterized an enzyme with epimerase activity, which might be responsible for the conversion of mannose into glucose. The role of the identified enzymes in the hydrolysis of galactomannan by aerobic bacteria is discussed.
  FEMS Microbiology Letters 06/2006; 261(1):123 - 132. · 2.04 Impact Factor
• Source

  Available from: ox.ac.uk

  Article: Structural dissection and high-throughput screening of mannosylglycerate synthase.

  James Flint, Edward Taylor, Min Yang, David N Bolam, Louise E Tailford, Carlos Martinez-Fleites, Eleanor J Dodson, Benjamin G Davis, Harry J Gilbert, Gideon J Davies
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  ABSTRACT: The enzymatic transfer of activated mannose yields mannosides in glycoconjugates and oligo- and polysaccharides. Yet, despite its biological necessity, the mechanism by which glycosyltransferases recognize mannose and catalyze its transfer to acceptor molecules is poorly understood. Here, we report broad high-throughput screening and kinetic analyses of both natural and synthetic substrates of Rhodothermus marinus mannosylglycerate synthase (MGS), which catalyzes the formation of the stress protectant 2-O-alpha-D-mannosyl glycerate. The sequence of MGS indicates that it is at the cusp of inverting and retaining transferases. The structures of apo MGS and complexes with donor and acceptor molecules, including GDP-mannose, combined with mutagenesis of the binding and catalytic sites, unveil the mannosyl transfer center. Nucleotide specificity is as important in GDP-D-mannose recognition as the nature of the donor sugar.
  Nature Structural &#38 Molecular Biology 08/2005; 12(7):608-14. · 12.71 Impact Factor
• Article: Insights into the molecular determinants of substrate specificity in glycoside hydrolase family 5 revealed by the crystal structure and kinetics of Cellvibrio mixtus mannosidase 5A.

  Fernando M V Dias, Florence Vincent, Gavin Pell, José A M Prates, Maria S J Centeno, Louise E Tailford, Luís M A Ferreira, Carlos M G A Fontes, Gideon J Davies, Harry J Gilbert
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  ABSTRACT: The enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze beta-mannans and beta-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 beta-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (-1) and two aglycone (+1 and +2) sugar-binding subsites. The -1 subsite displays absolute specificity for mannose, whereas the +1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The +2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 A crystal structure reveals that CmMan5A adopts a (beta/alpha)(8) barrel fold, and superimposition with GH5 endo-mannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand beta8 and helix alpha8 comprising residues 378-412. This insertion forms a "double" steric barrier, formed by two short beta-strands that function to "block" the substrate binding cleft at the edge of the -1 subsite forming the "exo" active center topology of CmMan5A.
  Journal of Biological Chemistry 07/2004; 279(24):25517-26. · 4.77 Impact Factor
• Article: Structural and biochemical evidence for a boat-like transition state in β-mannosidases

  Louise E Tailford, Wendy A Offen, Nicola L Smith, Claire Dumon, Carl Morland, Julie Gratien, Marie-Pierre Heck, Robert V Stick, Yves Blériot, Andrea Vasella, Harry J Gilbert, Gideon J Davies