Xylose Isomerase

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Dagmar Ringe - One of the best experts on this subject based on the ideXlab platform.

  • Xylose Isomerase in substrate and inhibitor michaelis states atomic resolution studies of a metal mediated hydride shift
    Biochemistry, 2004
    Co-Authors: Timothy D Fenn, Dagmar Ringe, Gregory A Petsko
    Abstract:

    Xylose Isomerase (E.C. 5.3.1.5) catalyzes the interconversion of aldose and ketose sugars and has an absolute requirement for two divalent cations at its active site to drive the hydride transfer rates of sugar isomerization. Evidence suggests some degree of metal movement at the second metal site, although how this movement may affect catalysis is unknown. The 0.95 A resolution structure of the xylitol-inhibited enzyme presented here suggests three alternative positions for the second metal ion, only one of which appears positioned in a catalytically competent manner. To complete the reaction, an active site hydroxyl species appears appropriately positioned for hydrogen transfer, as evidenced by precise bonding distances. Conversely, the 0.98 A resolution structure of the enzyme with glucose bound in the α-pyranose state only shows one of the metal ion conformations at the second metal ion binding site, suggesting that the linear form of the sugar is required to promote the second and third metal ion con...

  • design synthesis and characterization of a potent Xylose Isomerase inhibitor d threonohydroxamic acid and high resolution x ray crystallographic structure of the enzyme inhibitor complex
    Biochemistry, 1995
    Co-Authors: Karen N Allen, Gregory A Petsko, Arnon Lavie, Dagmar Ringe
    Abstract:

    : The binding of a potent inhibitor to the enzyme D-Xylose Isomerase from Streptomyces olivochromogenes was examined by kinetics and X-ray crystallography. The inhibitor D-threonohydroxamic acid (THA) was designed to mimic the putative transition state of the isomerization step catalyzed by the enzyme on the substrate Xylose. THA was synthesized and found to be a slow-binding competitive inhibitor with the substrate glucose. The Ki < or = 100 nM was at least one million-fold less than the KM for glucose. The X-ray crystallographic structure of Xylose Isomerase with THA soaked into the crystals (concentration = 1000Ki) was obtained to 1.6-A resolution and refined to an R factor of 21.6%. The free enzyme and the enzyme in the Xylose Isomerase-THA complex show no significant structural differences. THA binds in an analogous fashion to glucose, in a linear conformation, forming ligands with Mg-1 and Mg-2 and hydrogen bonds with His53 and Lys182. On the basis of these similarities to glucose binding and its potent inhibition, we propose that THA resembles the transition state for the enzyme-catalyzed hydride transfer reaction. The THA C2 hydroxyl forms a bridging ligand between Mg-1 and Mg-2; it must be deprotonated to do so. By analogy, we propose that, during the catalytic reaction, C2 of the substrate glucose is deprotonated, and that this proton can be moved to the C1 hydroxyl concomitant with hydride transfer. We find evidence for metal movement during catalysis upon deprotonation of the C2 hydroxyl, to allow formation of a bridging ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

  • design synthesis and characterization of a potent Xylose Isomerase inhibitor d threonohydroxamic acid and high resolution x ray crystallographic structure of the enzyme inhibitor complex
    Biochemistry, 1995
    Co-Authors: Karen N Allen, Gregory A Petsko, Arnon Lavie, Dagmar Ringe
    Abstract:

    : The binding of a potent inhibitor to the enzyme D-Xylose Isomerase from Streptomyces olivochromogenes was examined by kinetics and X-ray crystallography. The inhibitor D-threonohydroxamic acid (THA) was designed to mimic the putative transition state of the isomerization step catalyzed by the enzyme on the substrate Xylose. THA was synthesized and found to be a slow-binding competitive inhibitor with the substrate glucose. The Ki < or = 100 nM was at least one million-fold less than the KM for glucose. The X-ray crystallographic structure of Xylose Isomerase with THA soaked into the crystals (concentration = 1000Ki) was obtained to 1.6-A resolution and refined to an R factor of 21.6%. The free enzyme and the enzyme in the Xylose Isomerase-THA complex show no significant structural differences. THA binds in an analogous fashion to glucose, in a linear conformation, forming ligands with Mg-1 and Mg-2 and hydrogen bonds with His53 and Lys182. On the basis of these similarities to glucose binding and its potent inhibition, we propose that THA resembles the transition state for the enzyme-catalyzed hydride transfer reaction. The THA C2 hydroxyl forms a bridging ligand between Mg-1 and Mg-2; it must be deprotonated to do so. By analogy, we propose that, during the catalytic reaction, C2 of the substrate glucose is deprotonated, and that this proton can be moved to the C1 hydroxyl concomitant with hydride transfer. We find evidence for metal movement during catalysis upon deprotonation of the C2 hydroxyl, to allow formation of a bridging ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

  • x ray crystallographic structures of d Xylose Isomerase substrate complexes position the substrate and provide evidence for metal movement during catalysis
    Biochemistry, 1994
    Co-Authors: Arnon Lavie, Karen N Allen, Gregory A Petsko, Dagmar Ringe
    Abstract:

    The X-ray crystallographic structures of the metal-activated enzyme Xylose Isomerase from Streptomyces olivochromogenes with the substrates D-glucose, 3-O-methyl-D-glucose and in the absence of substrate were determined to 1.96-, 2.19-, and 1.81-A resolution and refined to R-factors of 16.6%, 15.9%, and 16.1%, respectively. Xylose Isomerase catalyzes the interconversion between glucose and fructose (Xylose and xylulose under physiological conditions) by utilizing two metal cofactors to promote a hydride shift; the metals are bridged by a glutamate residue. This puts Xylose Isomerase in the small but rapidly growing family of enzymes with a bridged bimetallic active site, in which both metals are involved in the chemical transformation. The substrate 3-O-methylglucose was chosen in order to position the glucose molecule in the observed electron density unambiguously. Of the two essential magnesium ions per active site, Mg-2 was observed to occupy two alternate positions, separated by 1.8 A, in the substrate-soaked structures. The deduced movement was not observed in the structure without substrate present and is attributed to a step following substrate binding but prior to isomerization. The substrates glucose and 3-O-methylglucose are observed in their linear extended forms and make identical interactions with the enzyme by forming ligands to Mg-1 through O2 and O4 and by forming hydrogen bonds with His53 through O5 and Lys182 through O1. Mg-2 has a water ligand that is interpreted in the crystal structure in the absence of substrate as a hydroxide ion and in the presence of substrate as a water molecule. This hydroxide ion may act as a base to deprotonate the glucose O2 and subsequently protonate the product fructose O1 concomitant with hydride transfer. Calculations of the solvent-accessible surface of possible dimers, with and without the alpha-helical C-terminal domain, suggest that the tetramer is the active form of this Xylose Isomerase.

  • isotopic exchange plus substrate and inhibition kinetics of d Xylose Isomerase do not support a proton transfer mechanism
    Biochemistry, 1994
    Co-Authors: Karen N Allen, Gregory A Petsko, Arnon Lavie, Gregory K. Farber, Arthur Glasfeld, Dagmar Ringe
    Abstract:

    The D-Xylose Isomerase of Streptomyces olivochromogenes is a Mg2+- or Mn(2+)-dependent enzyme that catalyzes the aldose-ketose isomerization of Xylose to xylulose or of glucose to fructose. Proton exchange into water during enzyme-catalyzed isomerization of C-2 tritiated glucose at 15, 25 and 55 degrees C shows < 0.6% exchange (the loss of one proton in every billion turnovers). High concentrations of guanidine hydrochloride and extremes of pH had no effect on the amount of exchange detected. Such a low percentage of exchange is inconsistent with a proton-transfer mechanism as the main kinetic pathway for isomerization. 19F NMR experiments showed no release of fluoride after incubation of the enzyme for 4 weeks with 800 mM 3-deoxy-3-fluoroglucose or 3-deoxy-3-fluoroallose (both are competitive inhibitors with Ki values of 600 mM). This result is also inconsistent with a proton-transfer mechanism. A hydride-shift mechanism following ring opening has been proposed for the isomerization. Enzyme-catalyzed ring opening was directly measured by demonstrating H2S release upon reaction of Xylose Isomerase with 1-thioglucose. D-Xylose Isomerase-catalyzed interconversion of glucose to fructose exhibited linear Arrhenius behavior with an activation energy of 14 kcal/mol from 0 to 50 degrees C. No change in rate-determining step occurs over this temperature range. 13C NMR experiments with glucose show that enzyme-bound magnesium or manganese does not interact specifically with any one site on the sugar. These results are consistent with nonproductive binding modes for the substrate glucose in addition to productive binding.

Jeffrey A. Mertens - One of the best experts on this subject based on the ideXlab platform.

  • growth and fermentation of d Xylose by saccharomyces cerevisiae expressing a novel d Xylose Isomerase originating from the bacterium prevotella ruminicola tc2 24
    Biotechnology for Biofuels, 2013
    Co-Authors: Ronald E. Hector, Michael A Cotta, Bruce S. Dien, Jeffrey A. Mertens
    Abstract:

    Background Saccharomyces cerevisiae strains expressing D-Xylose Isomerase (XI) produce some of the highest reported ethanol yields from D-Xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are either not functional, require additional strain modification, or have low affinity for D-Xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for D-Xylose fermentation.

  • growth and fermentation of d Xylose by saccharomyces cerevisiae expressing a novel d Xylose Isomerase originating from the bacterium prevotella ruminicola tc2 24
    Biotechnology for Biofuels, 2013
    Co-Authors: Ronald E. Hector, Michael A Cotta, Bruce S. Dien, Jeffrey A. Mertens
    Abstract:

    Saccharomyces cerevisiae strains expressing D-Xylose Isomerase (XI) produce some of the highest reported ethanol yields from D-Xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are either not functional, require additional strain modification, or have low affinity for D-Xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for D-Xylose fermentation. Four XIs originating from rumen and intestinal bacteria were isolated and expressed in a S. cerevisiae CEN.PK2-1C parental strain primed for D-Xylose metabolism by over expression of its native D-xylulokinase. Three of the XIs were functional in S. cerevisiae, based on the strain’s ability to grow in D-Xylose medium. The most promising strain, expressing the XI mined from Prevotella ruminicola TC2-24, was further adapted for aerobic and fermentative growth by serial transfers of D-Xylose cultures under aerobic, and followed by microaerobic conditions. The evolved strain had a specific growth rate of 0.23 h-1 on D-Xylose medium, which is comparable to the best reported results for analogous S. cerevisiae strains including those expressing the Piromyces sp. E2 XI. When used to ferment D-Xylose, the adapted strain produced 13.6 g/L ethanol in 91 h with a metabolic yield of 83% of theoretical. From analysis of the P. ruminicola XI, it was determined the enzyme possessed a V max of 0.81 μmole/min/mg protein and a K m of 34 mM. This study identifies a new Xylose Isomerase from the rumen bacterium Prevotella ruminicola TC2-24 that has one of the highest affinities and specific activities compared to other bacterial and fungal D-Xylose Isomerases expressed in yeast. When expressed in S. cerevisiae and used to ferment D-Xylose, very high ethanol yield was obtained. This new XI should be a promising resource for constructing other D-Xylose fermenting strains, including industrial yeast genetic backgrounds.

Matti Leisola - One of the best experts on this subject based on the ideXlab platform.

  • stochastic boundary molecular dynamics simulation of l ribose in the active site of actinoplanes missouriensis Xylose Isomerase and its val135asn mutant with improved reaction rate
    Biochimica et Biophysica Acta, 2005
    Co-Authors: Harri Santa, Matti Leisola, Ossi Pastinen, Johanna Karimaki, Juha Kammonen, Olli Lehtonen, Ossi Turunen
    Abstract:

    We used molecular dynamics simulations to study how a non-natural substrate, L-ribose, interacts with the active site of Actinoplanes missouriensis Xylose Isomerase. The simulations showed that L-ribose does not stay liganded in the active site in the same way as D-Xylose, in which the oxygens O2 and O4 are liganded to the metal M1. The oxygen O4 of L-ribose moved away from the metal M1 to an upside down position. Furthermore, the distances of the carbons C1 and C2 of L-ribose to the catalytic metal M2 were higher than in the case of D-Xylose. These findings explain the extremely low reaction rate of Xylose Isomerase with L-ribose. The mutation V135N close to the C5-OH of the substrate increased the reaction efficiency 2- to 4-fold with L-ribose. V135N did not affect the reaction with D-Xylose and L-arabinose, whereas the reaction with D-glucose was impaired, probably due to a hydrogen bond between Asn-135 and the substrate. When L-ribose was the substrate, Asn-135 formed a hydrogen bond to Glu-181. As a consequence, O4 of L-ribose stayed liganded to the metal M1 in the V135N mutant in molecular dynamics simulations. This explains the decreased K(m) of the V135N mutant with L-ribose.

  • engineering the substrate specificity of Xylose Isomerase
    Protein Engineering Design & Selection, 2005
    Co-Authors: Johanna Karimaki, Matti Leisola, Ossi Pastinen, Tarja Parkkinen, Harri Santa, Juha Rouvinen, Ossi Turunen
    Abstract:

    Xylose Isomerase (XI) catalyzes the isomerization and epimerization of hexoses, pentoses and tetroses. In order to clarify the reasons for the low reaction efficiency of a pentose sugar, L-arabinose, we determined the crystal structure of Streptomyces rubiginosus XI complexed with L-arabinose. The crystal structure revealed that, when compared with D-Xylose and D-glucose, L-arabinose binds to the active site in a partially different position, in which the ligand has difficulties in binding the catalytic metal M2. Lys183 has been thought to stabilize the open substrate conformation by hydrogen bonding to oxygen O1. Our results with L-arabinose showed that the substrate stays in a linear form even without a hydrogen bond between Lys183 and oxygen O1. We engineered mutations to the active site of Actinoplanes missouriensis XI to improve the reaction efficiency with L-arabinose. The mutation F26W was intended to shift the position of oxygen O1 of L-arabinose closer to the catalytic metal M2. This effect of F26W was modeled by free energy perturbation simulations. In line with this, F26W increased 2-fold the catalytic efficiency of XI with L-arabinose; the increase was seen mainly in kcat. The mutation Q256D was outside the sphere of the catalytic residues and probably modified the electrostatic properties of the active site. It improved 3-fold the catalytic efficiency of XI with L-arabinose; this increase was seen in both Km and kcat. This study showed that it is possible to engineer the substrate specificity of XI.

  • chromatographic separation of nucleosides using a cross linked Xylose Isomerase crystal stationary phase
    IEEE Journal of Solid-state Circuits, 2004
    Co-Authors: Jouni Jokela, Matti Leisola
    Abstract:

    Cross-linked Xylose Isomerase (EC 5.3.1.5., from Streptomyces rubiginosus) crystals (CLXIC) packed into a 7.8×300 mm steel column showed specific affinity towards uridine (Urd), cytidine (Cyd), adenosine (Ado), guanosine (Guo), and thymidine. These nucleosides eluted out of the CLXIC column in the same order as the corresponding nucleoside bases, indicating that the retention depends mainly on the base component of the molecule. The interaction of nucleosides with the CLXIC material was not based merely on ion exchange or hydrophobic interactions but also on the unique properties of the CLXIC column. Decrease in temperature increased the retention but not the resolution factors of the adjacent nucleosides. The CLXIC column maintained its separation capacity even when 100 mg of ribonucleosides in equimass amounts were injected into the column in a volume of 1 mL corresponding to 10% of the total column volume. Analysis of sugar beet molasses, a side stream from sucrose production, showed it to contain 1–2.5 mg mL–1 of Urd, Cyd, Ado, and Guo. The CLXIC column was able to separate and enrich these nucleosides also from highly viscous sugar beet molasses. The CLXIC column was especially efficient in the purification of guanosine. Other commercially interesting sugar beet molasses components such as the acidic compounds betaine, γ-amino butyric acid, and D- and L-pyroglutamic acids or neutral sucrose did not interact with the CLXIC material.

  • enhanced glucose to fructose conversion in acetone with Xylose Isomerase stabilized by crystallization and cross linking
    Biotechnology Progress, 2004
    Co-Authors: Kati Vilonen, Jouni Jokela, Matti Leisola, Antti Vuolanto, Outi A I Krause
    Abstract:

    The effects of acetone and ethanol on glucose to fructose conversion catalyzed by soluble and cross-linked crystalline (CLXIC) Xylose Isomerase were studied. Relative to pure buffer solvent, the fructose production rate was more than doubled in 50% acetone. The same kind of increase in the isomerization rate was not seen with ethanol. Increase both in acetone and in ethanol concentration in the reaction solvent enhanced the production of fructose. At 50 degrees C in pure buffer solvent the reaction mixture contained 49% fructose in equilibrium and in 90% acetone the fructose equilibrium content was 64%. Furthermore, CLXIC was relatively stable in the presence of high concentration of acetone: 70-80% of activity was left after incubation for 24 h at 50 degrees C in buffer solutions (pH 7.2) containing 10-90% acetone. In buffer containing 50% ethanol only 2% of the initial activity of CLXIC was retained after 24 h at 50 degrees C. Soluble Xylose Isomerase was considerably less stable than CLXIC in both acetone- and ethanol-containing solutions. These results show that the addition of acetone enhances the production of fructose from glucose by enhancing the reaction rate and shifting the equilibrium toward fructose. However, Xylose Isomerase must be in the form of cross-linked crystals for maximal activity and stability.

  • solubility and crystallization of Xylose Isomerase from streptomyces rubiginosus
    Journal of Crystal Growth, 2003
    Co-Authors: Antti Vuolanto, Matti Leisola, Sinikka Uotila, Kalevi Visuri
    Abstract:

    Abstract We have studied the crystallization and crystal solubility of Xylose Isomerase (XI) from Streptomyces rubiginosus . In this paper, we show a rational approach for developing a large-scale crystallization process for XI. Firstly, we measured the crystal solubility in salt solutions with respect to salt concentration, temperature and pH. In ammonium sulfate the solubility of XI decreased logarithmically when increasing the salt concentration. Surprisingly, the XI crystals had a solubility minimum at low concentration of magnesium sulfate. The solubility of XI in 0.17 M magnesium sulfate was less than 0.5 g l −1 . The solubility of XI increased logarithmically when increasing the temperature. We also found a solubility minimum around pH 7. This is far from the isoelectric point of XI (pH 3.95). Secondly, based on the solubility study, we developed a large-scale crystallization process for XI. In a simple and economical cooling crystallization of XI from 0.17 M magnesium sulfate solution, the recovery of crystalline active enzyme was over 95%. Moreover, we developed a process for production of uniform crystals and produced homogenous crystals with average crystal sizes between 12 and 360 μm.

Gregory A Petsko - One of the best experts on this subject based on the ideXlab platform.

  • Xylose Isomerase in substrate and inhibitor michaelis states atomic resolution studies of a metal mediated hydride shift
    Biochemistry, 2004
    Co-Authors: Timothy D Fenn, Dagmar Ringe, Gregory A Petsko
    Abstract:

    Xylose Isomerase (E.C. 5.3.1.5) catalyzes the interconversion of aldose and ketose sugars and has an absolute requirement for two divalent cations at its active site to drive the hydride transfer rates of sugar isomerization. Evidence suggests some degree of metal movement at the second metal site, although how this movement may affect catalysis is unknown. The 0.95 A resolution structure of the xylitol-inhibited enzyme presented here suggests three alternative positions for the second metal ion, only one of which appears positioned in a catalytically competent manner. To complete the reaction, an active site hydroxyl species appears appropriately positioned for hydrogen transfer, as evidenced by precise bonding distances. Conversely, the 0.98 A resolution structure of the enzyme with glucose bound in the α-pyranose state only shows one of the metal ion conformations at the second metal ion binding site, suggesting that the linear form of the sugar is required to promote the second and third metal ion con...

  • design synthesis and characterization of a potent Xylose Isomerase inhibitor d threonohydroxamic acid and high resolution x ray crystallographic structure of the enzyme inhibitor complex
    Biochemistry, 1995
    Co-Authors: Karen N Allen, Gregory A Petsko, Arnon Lavie, Dagmar Ringe
    Abstract:

    : The binding of a potent inhibitor to the enzyme D-Xylose Isomerase from Streptomyces olivochromogenes was examined by kinetics and X-ray crystallography. The inhibitor D-threonohydroxamic acid (THA) was designed to mimic the putative transition state of the isomerization step catalyzed by the enzyme on the substrate Xylose. THA was synthesized and found to be a slow-binding competitive inhibitor with the substrate glucose. The Ki < or = 100 nM was at least one million-fold less than the KM for glucose. The X-ray crystallographic structure of Xylose Isomerase with THA soaked into the crystals (concentration = 1000Ki) was obtained to 1.6-A resolution and refined to an R factor of 21.6%. The free enzyme and the enzyme in the Xylose Isomerase-THA complex show no significant structural differences. THA binds in an analogous fashion to glucose, in a linear conformation, forming ligands with Mg-1 and Mg-2 and hydrogen bonds with His53 and Lys182. On the basis of these similarities to glucose binding and its potent inhibition, we propose that THA resembles the transition state for the enzyme-catalyzed hydride transfer reaction. The THA C2 hydroxyl forms a bridging ligand between Mg-1 and Mg-2; it must be deprotonated to do so. By analogy, we propose that, during the catalytic reaction, C2 of the substrate glucose is deprotonated, and that this proton can be moved to the C1 hydroxyl concomitant with hydride transfer. We find evidence for metal movement during catalysis upon deprotonation of the C2 hydroxyl, to allow formation of a bridging ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

  • design synthesis and characterization of a potent Xylose Isomerase inhibitor d threonohydroxamic acid and high resolution x ray crystallographic structure of the enzyme inhibitor complex
    Biochemistry, 1995
    Co-Authors: Karen N Allen, Gregory A Petsko, Arnon Lavie, Dagmar Ringe
    Abstract:

    : The binding of a potent inhibitor to the enzyme D-Xylose Isomerase from Streptomyces olivochromogenes was examined by kinetics and X-ray crystallography. The inhibitor D-threonohydroxamic acid (THA) was designed to mimic the putative transition state of the isomerization step catalyzed by the enzyme on the substrate Xylose. THA was synthesized and found to be a slow-binding competitive inhibitor with the substrate glucose. The Ki < or = 100 nM was at least one million-fold less than the KM for glucose. The X-ray crystallographic structure of Xylose Isomerase with THA soaked into the crystals (concentration = 1000Ki) was obtained to 1.6-A resolution and refined to an R factor of 21.6%. The free enzyme and the enzyme in the Xylose Isomerase-THA complex show no significant structural differences. THA binds in an analogous fashion to glucose, in a linear conformation, forming ligands with Mg-1 and Mg-2 and hydrogen bonds with His53 and Lys182. On the basis of these similarities to glucose binding and its potent inhibition, we propose that THA resembles the transition state for the enzyme-catalyzed hydride transfer reaction. The THA C2 hydroxyl forms a bridging ligand between Mg-1 and Mg-2; it must be deprotonated to do so. By analogy, we propose that, during the catalytic reaction, C2 of the substrate glucose is deprotonated, and that this proton can be moved to the C1 hydroxyl concomitant with hydride transfer. We find evidence for metal movement during catalysis upon deprotonation of the C2 hydroxyl, to allow formation of a bridging ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

  • x ray crystallographic structures of d Xylose Isomerase substrate complexes position the substrate and provide evidence for metal movement during catalysis
    Biochemistry, 1994
    Co-Authors: Arnon Lavie, Karen N Allen, Gregory A Petsko, Dagmar Ringe
    Abstract:

    The X-ray crystallographic structures of the metal-activated enzyme Xylose Isomerase from Streptomyces olivochromogenes with the substrates D-glucose, 3-O-methyl-D-glucose and in the absence of substrate were determined to 1.96-, 2.19-, and 1.81-A resolution and refined to R-factors of 16.6%, 15.9%, and 16.1%, respectively. Xylose Isomerase catalyzes the interconversion between glucose and fructose (Xylose and xylulose under physiological conditions) by utilizing two metal cofactors to promote a hydride shift; the metals are bridged by a glutamate residue. This puts Xylose Isomerase in the small but rapidly growing family of enzymes with a bridged bimetallic active site, in which both metals are involved in the chemical transformation. The substrate 3-O-methylglucose was chosen in order to position the glucose molecule in the observed electron density unambiguously. Of the two essential magnesium ions per active site, Mg-2 was observed to occupy two alternate positions, separated by 1.8 A, in the substrate-soaked structures. The deduced movement was not observed in the structure without substrate present and is attributed to a step following substrate binding but prior to isomerization. The substrates glucose and 3-O-methylglucose are observed in their linear extended forms and make identical interactions with the enzyme by forming ligands to Mg-1 through O2 and O4 and by forming hydrogen bonds with His53 through O5 and Lys182 through O1. Mg-2 has a water ligand that is interpreted in the crystal structure in the absence of substrate as a hydroxide ion and in the presence of substrate as a water molecule. This hydroxide ion may act as a base to deprotonate the glucose O2 and subsequently protonate the product fructose O1 concomitant with hydride transfer. Calculations of the solvent-accessible surface of possible dimers, with and without the alpha-helical C-terminal domain, suggest that the tetramer is the active form of this Xylose Isomerase.

  • isotopic exchange plus substrate and inhibition kinetics of d Xylose Isomerase do not support a proton transfer mechanism
    Biochemistry, 1994
    Co-Authors: Karen N Allen, Gregory A Petsko, Arnon Lavie, Gregory K. Farber, Arthur Glasfeld, Dagmar Ringe
    Abstract:

    The D-Xylose Isomerase of Streptomyces olivochromogenes is a Mg2+- or Mn(2+)-dependent enzyme that catalyzes the aldose-ketose isomerization of Xylose to xylulose or of glucose to fructose. Proton exchange into water during enzyme-catalyzed isomerization of C-2 tritiated glucose at 15, 25 and 55 degrees C shows < 0.6% exchange (the loss of one proton in every billion turnovers). High concentrations of guanidine hydrochloride and extremes of pH had no effect on the amount of exchange detected. Such a low percentage of exchange is inconsistent with a proton-transfer mechanism as the main kinetic pathway for isomerization. 19F NMR experiments showed no release of fluoride after incubation of the enzyme for 4 weeks with 800 mM 3-deoxy-3-fluoroglucose or 3-deoxy-3-fluoroallose (both are competitive inhibitors with Ki values of 600 mM). This result is also inconsistent with a proton-transfer mechanism. A hydride-shift mechanism following ring opening has been proposed for the isomerization. Enzyme-catalyzed ring opening was directly measured by demonstrating H2S release upon reaction of Xylose Isomerase with 1-thioglucose. D-Xylose Isomerase-catalyzed interconversion of glucose to fructose exhibited linear Arrhenius behavior with an activation energy of 14 kcal/mol from 0 to 50 degrees C. No change in rate-determining step occurs over this temperature range. 13C NMR experiments with glucose show that enzyme-bound magnesium or manganese does not interact specifically with any one site on the sugar. These results are consistent with nonproductive binding modes for the substrate glucose in addition to productive binding.

Karen N Allen - One of the best experts on this subject based on the ideXlab platform.

  • binding energy and catalysis by d Xylose Isomerase kinetic product and x ray crystallographic analysis of enzyme catalyzed isomerization of r glyceraldehyde
    Biochemistry, 2011
    Co-Authors: Maria M Toteva, Karen N Allen, Nicholas R Silvaggi, John P Richard
    Abstract:

    d-Xylose Isomerase (XI) and triosephosphate Isomerase (TIM) catalyze the aldose–ketose isomerization reactions of d-Xylose and d-glyceraldehyde 3-phosphate (DGAP), respectively. d-Glyceraldehyde (DGA) is the triose fragment common to the substrates for XI and TIM. The XI-catalyzed isomerization of DGA to give dihydroxyacetone (DHA) in D2O was monitored by 1H nuclear magnetic resonance spectroscopy, and a kcat/Km of 0.034 M–1 s–1 was determined for this isomerization at pD 7.0. This is similar to the kcat/Km of 0.017 M–1 s–1 for the TIM-catalyzed carbon deprotonation reaction of DGA in D2O at pD 7.0 [Amyes, T. L., O’Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325–11326]. The much larger activation barrier for XI-catalyzed isomerization of d-Xylose (kcat/Km = 490 M–1 s–1) versus that for the TIM-catalyzed isomerization of DGAP (kcat/Km = 9.6 × 106 M–1 s–1) is due to (i) the barrier to conversion of cyclic d-Xylose to the reactive linear sugar (5.4 kcal/mol) being larger than that for...

  • design synthesis and characterization of a potent Xylose Isomerase inhibitor d threonohydroxamic acid and high resolution x ray crystallographic structure of the enzyme inhibitor complex
    Biochemistry, 1995
    Co-Authors: Karen N Allen, Gregory A Petsko, Arnon Lavie, Dagmar Ringe
    Abstract:

    : The binding of a potent inhibitor to the enzyme D-Xylose Isomerase from Streptomyces olivochromogenes was examined by kinetics and X-ray crystallography. The inhibitor D-threonohydroxamic acid (THA) was designed to mimic the putative transition state of the isomerization step catalyzed by the enzyme on the substrate Xylose. THA was synthesized and found to be a slow-binding competitive inhibitor with the substrate glucose. The Ki < or = 100 nM was at least one million-fold less than the KM for glucose. The X-ray crystallographic structure of Xylose Isomerase with THA soaked into the crystals (concentration = 1000Ki) was obtained to 1.6-A resolution and refined to an R factor of 21.6%. The free enzyme and the enzyme in the Xylose Isomerase-THA complex show no significant structural differences. THA binds in an analogous fashion to glucose, in a linear conformation, forming ligands with Mg-1 and Mg-2 and hydrogen bonds with His53 and Lys182. On the basis of these similarities to glucose binding and its potent inhibition, we propose that THA resembles the transition state for the enzyme-catalyzed hydride transfer reaction. The THA C2 hydroxyl forms a bridging ligand between Mg-1 and Mg-2; it must be deprotonated to do so. By analogy, we propose that, during the catalytic reaction, C2 of the substrate glucose is deprotonated, and that this proton can be moved to the C1 hydroxyl concomitant with hydride transfer. We find evidence for metal movement during catalysis upon deprotonation of the C2 hydroxyl, to allow formation of a bridging ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

  • design synthesis and characterization of a potent Xylose Isomerase inhibitor d threonohydroxamic acid and high resolution x ray crystallographic structure of the enzyme inhibitor complex
    Biochemistry, 1995
    Co-Authors: Karen N Allen, Gregory A Petsko, Arnon Lavie, Dagmar Ringe
    Abstract:

    : The binding of a potent inhibitor to the enzyme D-Xylose Isomerase from Streptomyces olivochromogenes was examined by kinetics and X-ray crystallography. The inhibitor D-threonohydroxamic acid (THA) was designed to mimic the putative transition state of the isomerization step catalyzed by the enzyme on the substrate Xylose. THA was synthesized and found to be a slow-binding competitive inhibitor with the substrate glucose. The Ki < or = 100 nM was at least one million-fold less than the KM for glucose. The X-ray crystallographic structure of Xylose Isomerase with THA soaked into the crystals (concentration = 1000Ki) was obtained to 1.6-A resolution and refined to an R factor of 21.6%. The free enzyme and the enzyme in the Xylose Isomerase-THA complex show no significant structural differences. THA binds in an analogous fashion to glucose, in a linear conformation, forming ligands with Mg-1 and Mg-2 and hydrogen bonds with His53 and Lys182. On the basis of these similarities to glucose binding and its potent inhibition, we propose that THA resembles the transition state for the enzyme-catalyzed hydride transfer reaction. The THA C2 hydroxyl forms a bridging ligand between Mg-1 and Mg-2; it must be deprotonated to do so. By analogy, we propose that, during the catalytic reaction, C2 of the substrate glucose is deprotonated, and that this proton can be moved to the C1 hydroxyl concomitant with hydride transfer. We find evidence for metal movement during catalysis upon deprotonation of the C2 hydroxyl, to allow formation of a bridging ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

  • x ray crystallographic structures of d Xylose Isomerase substrate complexes position the substrate and provide evidence for metal movement during catalysis
    Biochemistry, 1994
    Co-Authors: Arnon Lavie, Karen N Allen, Gregory A Petsko, Dagmar Ringe
    Abstract:

    The X-ray crystallographic structures of the metal-activated enzyme Xylose Isomerase from Streptomyces olivochromogenes with the substrates D-glucose, 3-O-methyl-D-glucose and in the absence of substrate were determined to 1.96-, 2.19-, and 1.81-A resolution and refined to R-factors of 16.6%, 15.9%, and 16.1%, respectively. Xylose Isomerase catalyzes the interconversion between glucose and fructose (Xylose and xylulose under physiological conditions) by utilizing two metal cofactors to promote a hydride shift; the metals are bridged by a glutamate residue. This puts Xylose Isomerase in the small but rapidly growing family of enzymes with a bridged bimetallic active site, in which both metals are involved in the chemical transformation. The substrate 3-O-methylglucose was chosen in order to position the glucose molecule in the observed electron density unambiguously. Of the two essential magnesium ions per active site, Mg-2 was observed to occupy two alternate positions, separated by 1.8 A, in the substrate-soaked structures. The deduced movement was not observed in the structure without substrate present and is attributed to a step following substrate binding but prior to isomerization. The substrates glucose and 3-O-methylglucose are observed in their linear extended forms and make identical interactions with the enzyme by forming ligands to Mg-1 through O2 and O4 and by forming hydrogen bonds with His53 through O5 and Lys182 through O1. Mg-2 has a water ligand that is interpreted in the crystal structure in the absence of substrate as a hydroxide ion and in the presence of substrate as a water molecule. This hydroxide ion may act as a base to deprotonate the glucose O2 and subsequently protonate the product fructose O1 concomitant with hydride transfer. Calculations of the solvent-accessible surface of possible dimers, with and without the alpha-helical C-terminal domain, suggest that the tetramer is the active form of this Xylose Isomerase.

  • isotopic exchange plus substrate and inhibition kinetics of d Xylose Isomerase do not support a proton transfer mechanism
    Biochemistry, 1994
    Co-Authors: Karen N Allen, Gregory A Petsko, Arnon Lavie, Gregory K. Farber, Arthur Glasfeld, Dagmar Ringe
    Abstract:

    The D-Xylose Isomerase of Streptomyces olivochromogenes is a Mg2+- or Mn(2+)-dependent enzyme that catalyzes the aldose-ketose isomerization of Xylose to xylulose or of glucose to fructose. Proton exchange into water during enzyme-catalyzed isomerization of C-2 tritiated glucose at 15, 25 and 55 degrees C shows < 0.6% exchange (the loss of one proton in every billion turnovers). High concentrations of guanidine hydrochloride and extremes of pH had no effect on the amount of exchange detected. Such a low percentage of exchange is inconsistent with a proton-transfer mechanism as the main kinetic pathway for isomerization. 19F NMR experiments showed no release of fluoride after incubation of the enzyme for 4 weeks with 800 mM 3-deoxy-3-fluoroglucose or 3-deoxy-3-fluoroallose (both are competitive inhibitors with Ki values of 600 mM). This result is also inconsistent with a proton-transfer mechanism. A hydride-shift mechanism following ring opening has been proposed for the isomerization. Enzyme-catalyzed ring opening was directly measured by demonstrating H2S release upon reaction of Xylose Isomerase with 1-thioglucose. D-Xylose Isomerase-catalyzed interconversion of glucose to fructose exhibited linear Arrhenius behavior with an activation energy of 14 kcal/mol from 0 to 50 degrees C. No change in rate-determining step occurs over this temperature range. 13C NMR experiments with glucose show that enzyme-bound magnesium or manganese does not interact specifically with any one site on the sugar. These results are consistent with nonproductive binding modes for the substrate glucose in addition to productive binding.