Metal ion roles and the movement of hydrogen during reaction catalyzed by D-xylose isomerase: a joint x-ray and neutron diffraction study

Abstract

Conversion of aldo to keto sugars by the metalloenzyme D-xylose isomerase (XI) is a multistep reaction that involves hydrogen transfer. We have determined the structure of this enzyme by neutron diffraction in order to locate H atoms (or their isotope D). Two studies are presented, one of XI containing cadmium and cyclic D-glucose (before sugar ring opening has occurred), and the other containing nickel and linear D-glucose (after ring opening has occurred but before isomerization). Previously we reported the neutron structures of ligand-free enzyme and enzyme with bound product. The data show that His54 is doubly protonated on the ring N in all four structures. Lys289 is neutral before ring opening and gains a proton after this; the catalytic metal-bound water is deprotonated to hydroxyl during isomerization and O5 is deprotonated. These results lead to new suggestions as to how changes might take place over the course of the reaction.

Figure 1

Active site of native XI in XI-Co_n. The residues coordinating metal co-factors, represented as magenta spheres, and those lining the cavity – Trp16, His54, Phe94, Trp137 and Phe26′ are shown. Phe26′ belongs to a symmetry-related subunit.

Figure 2

Cyclic glucose substrate binding in XI-Cd_CyclicSugar_n and comparison with XI-Co_n. Metal coordination to D₂O^cat and cyclic glucose is indicated by blue dotted lines; hydrogen bonds are shown as orange dashed lines and the O…D distances are in Å. (a) Superpositions of water structures in XI-Cd-CyclicSugar_n (colored by atom type) and XI-Co_n (colored magenta) complexes. (b) Superposition of selected active-site residues and water molecules in XI-Cd-CyclicSugar_n (atom-type colored sticks and violet spheres for Cd(II) cations) and XI-Co_n (dark green sticks). Neutron scattering density shown for XI-Cd-CyclicSugar_n is contoured at 1.6σ level. His54 and Lys183 are protonated, while Lys289 is neutral in both structures. One of the two distinct conformations of Lys289’s side chain is shown for XI-Co_n for clarity. Four water molecules are displaced by the cycle of per-deuterated glucose substrate. The catalytic water, marked D₂O^cat, retains its position and coordination to M2 in XI-Cd-CyclicSugar_n relative to XI-Co_n. (c) 2F_O-F_C neutron scattering (green, contoured at 1.6σ) and electron density (red, contoured at 2σ for light atoms and 10σ for Cd) maps for the cyclic per-deuterated glucose coordinated to Cd cations in XI-Cd-CyclicSugar_n.

Figure 2

Cyclic glucose substrate binding in XI-Cd_CyclicSugar_n and comparison with XI-Co_n. Metal coordination to D₂O^cat and cyclic glucose is indicated by blue dotted lines; hydrogen bonds are shown as orange dashed lines and the O…D distances are in Å. (a) Superpositions of water structures in XI-Cd-CyclicSugar_n (colored by atom type) and XI-Co_n (colored magenta) complexes. (b) Superposition of selected active-site residues and water molecules in XI-Cd-CyclicSugar_n (atom-type colored sticks and violet spheres for Cd(II) cations) and XI-Co_n (dark green sticks). Neutron scattering density shown for XI-Cd-CyclicSugar_n is contoured at 1.6σ level. His54 and Lys183 are protonated, while Lys289 is neutral in both structures. One of the two distinct conformations of Lys289’s side chain is shown for XI-Co_n for clarity. Four water molecules are displaced by the cycle of per-deuterated glucose substrate. The catalytic water, marked D₂O^cat, retains its position and coordination to M2 in XI-Cd-CyclicSugar_n relative to XI-Co_n. (c) 2F_O-F_C neutron scattering (green, contoured at 1.6σ) and electron density (red, contoured at 2σ for light atoms and 10σ for Cd) maps for the cyclic per-deuterated glucose coordinated to Cd cations in XI-Cd-CyclicSugar_n.

Figure 2

Cyclic glucose substrate binding in XI-Cd_CyclicSugar_n and comparison with XI-Co_n. Metal coordination to D₂O^cat and cyclic glucose is indicated by blue dotted lines; hydrogen bonds are shown as orange dashed lines and the O…D distances are in Å. (a) Superpositions of water structures in XI-Cd-CyclicSugar_n (colored by atom type) and XI-Co_n (colored magenta) complexes. (b) Superposition of selected active-site residues and water molecules in XI-Cd-CyclicSugar_n (atom-type colored sticks and violet spheres for Cd(II) cations) and XI-Co_n (dark green sticks). Neutron scattering density shown for XI-Cd-CyclicSugar_n is contoured at 1.6σ level. His54 and Lys183 are protonated, while Lys289 is neutral in both structures. One of the two distinct conformations of Lys289’s side chain is shown for XI-Co_n for clarity. Four water molecules are displaced by the cycle of per-deuterated glucose substrate. The catalytic water, marked D₂O^cat, retains its position and coordination to M2 in XI-Cd-CyclicSugar_n relative to XI-Co_n. (c) 2F_O-F_C neutron scattering (green, contoured at 1.6σ) and electron density (red, contoured at 2σ for light atoms and 10σ for Cd) maps for the cyclic per-deuterated glucose coordinated to Cd cations in XI-Cd-CyclicSugar_n.

Figure 3

Linear glucose substrate binding in XI-Ni_LinearSugar_n and comparison with XI-Cd_CyclicSugar_n. Metal coordination to D₂O^cat and cyclic glucose is indicated by blue dotted lines; hydrogen bonds are shown as orange dashed lines and the O…D distances are in Å. (a) Superpositions of substrates and water structures in XI-Ni-LinearSugar_n and XI-Cd-CyclicSugar_n complexes. (b) Superposition of selected active-site residues and water molecules in XI-Ni-LinearSugar_n (atom-type colored sticks and orange spheres for Ni(II) cations) and XI-Cd-CyclicSugar_n (dark green sticks). Neutron scattering density contoured at 1.6 level is shown for XI-Ni-LinearSugar_n, in which His54, Lys183 and Lys289 are protonated, while Lys289 is neutral and rotated away from Asp257 in XI-Cd-CyclicSugar_n. Carbon atoms C3, C4, C5 and C6 of the linear and cyclic per-deuterated glucose superimpose well, whereas C2 and C1 are rotated toward the catalytic metal M2. M2 has two distinct positions M2a and M2b 1.9 Å apart, with M2b coordinating O1 and O2 of the linear intermediate. The catalytic water marked D₂O^cat retains its position in XI-Ni-LinearSugar_n relative to XI-Cd-CyclicSugar_n. (c) 2F_O-F_C neutron scattering (green, contoured at 1.4σ) and electron density (red, contoured at 1.4σ for light atoms and 6σ for Ni) maps for the linear per-deuterated glucose coordinated to Ni cations in XI-Ni-LinearSugar_n.

Figure 3

Linear glucose substrate binding in XI-Ni_LinearSugar_n and comparison with XI-Cd_CyclicSugar_n. Metal coordination to D₂O^cat and cyclic glucose is indicated by blue dotted lines; hydrogen bonds are shown as orange dashed lines and the O…D distances are in Å. (a) Superpositions of substrates and water structures in XI-Ni-LinearSugar_n and XI-Cd-CyclicSugar_n complexes. (b) Superposition of selected active-site residues and water molecules in XI-Ni-LinearSugar_n (atom-type colored sticks and orange spheres for Ni(II) cations) and XI-Cd-CyclicSugar_n (dark green sticks). Neutron scattering density contoured at 1.6 level is shown for XI-Ni-LinearSugar_n, in which His54, Lys183 and Lys289 are protonated, while Lys289 is neutral and rotated away from Asp257 in XI-Cd-CyclicSugar_n. Carbon atoms C3, C4, C5 and C6 of the linear and cyclic per-deuterated glucose superimpose well, whereas C2 and C1 are rotated toward the catalytic metal M2. M2 has two distinct positions M2a and M2b 1.9 Å apart, with M2b coordinating O1 and O2 of the linear intermediate. The catalytic water marked D₂O^cat retains its position in XI-Ni-LinearSugar_n relative to XI-Cd-CyclicSugar_n. (c) 2F_O-F_C neutron scattering (green, contoured at 1.4σ) and electron density (red, contoured at 1.4σ for light atoms and 6σ for Ni) maps for the linear per-deuterated glucose coordinated to Ni cations in XI-Ni-LinearSugar_n.

Figure 3

Linear glucose substrate binding in XI-Ni_LinearSugar_n and comparison with XI-Cd_CyclicSugar_n. Metal coordination to D₂O^cat and cyclic glucose is indicated by blue dotted lines; hydrogen bonds are shown as orange dashed lines and the O…D distances are in Å. (a) Superpositions of substrates and water structures in XI-Ni-LinearSugar_n and XI-Cd-CyclicSugar_n complexes. (b) Superposition of selected active-site residues and water molecules in XI-Ni-LinearSugar_n (atom-type colored sticks and orange spheres for Ni(II) cations) and XI-Cd-CyclicSugar_n (dark green sticks). Neutron scattering density contoured at 1.6 level is shown for XI-Ni-LinearSugar_n, in which His54, Lys183 and Lys289 are protonated, while Lys289 is neutral and rotated away from Asp257 in XI-Cd-CyclicSugar_n. Carbon atoms C3, C4, C5 and C6 of the linear and cyclic per-deuterated glucose superimpose well, whereas C2 and C1 are rotated toward the catalytic metal M2. M2 has two distinct positions M2a and M2b 1.9 Å apart, with M2b coordinating O1 and O2 of the linear intermediate. The catalytic water marked D₂O^cat retains its position in XI-Ni-LinearSugar_n relative to XI-Cd-CyclicSugar_n. (c) 2F_O-F_C neutron scattering (green, contoured at 1.4σ) and electron density (red, contoured at 1.4σ for light atoms and 6σ for Ni) maps for the linear per-deuterated glucose coordinated to Ni cations in XI-Ni-LinearSugar_n.

Figure 4

Linear xylulose product binding in XI-Mg-Product_n and comparison with XI-Ni-LinearSugar_n. Hydrogen bonds are shown as orange dashed lines and the O…D distances are in Å. Superposition of selected active-site residues and reactive water species in XI-Mg-Product_n (atom-type colored sticks and magenta spheres for Mg(II) cations) and XI-Ni-LinearSugar_n (dark green sticks and spheres). Neutron scattering density contoured at 1.2σ level is shown for XI-Mg-Product_n, in which His54, Lys183 and Lys289 are protonated and hydrogen bonded to O5, O1 and Asp257, respectively. Linear per-deuterated xylulose and glucose superimpose well, except for the C2(O2)-C1(O1) portions of the molecules. The catalytic water (D₂O^cat) in XI-Ni-LinearSugar_n and deuteroxide (OD⁻) generated from it during the isomerization reaction in XI-Mg-Product_n occupy same position.

Figure 5

Lys289-Asp257 diad. Superposition of Asp257-Lys289 diad in the four neutron structures: XI-Co_n’s residues are colored by atom type (deuterium atoms are off-white); XI-Cd-CyclicSugar_n – magenta; XI-Ni-LinearSugar_n – salmon-red; XI-Mg-Product_n – grey.

Figure 6

A suggested mechanism for sugar interconversion reaction catalyzed by XI.

Scheme 1

D-glucose-to-D-fructose interconversion reaction. The reaction catalyzed by XI has been represented as consisting of three major steps (1) ring opening (2) isomerization (3) ring closure.