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ABSTRACT: d-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni 2+ cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg 2+ ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni 2+ ions occupying the catalytic metal site (M2) were found at two locations, while Mg 2+ in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH. PDB References: D-xylose isomerase, complex with Mn 2+ and xylitol, 4duo; complex with Ni 2+ and sorbitol, 4dvo.
Acta Crystallographica Section D Biological Crystallography 09/2012; 68(9):1201-1206. · 12.62 Impact Factor
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ABSTRACT: The Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. Neutron diffraction is a powerful technique for locating H atoms and can therefore provide unique information about how biological macromolecules function and interact with each other and smaller molecules. Users of the PCS have access to neutron beam time, deuteration facilities, the expression of proteins and the synthesis of substrates with stable isotopes and also support for data reduction and structure analysis. The beamline exploits the pulsed nature of spallation neutrons and a large electronic detector in order to collect wavelength-resolved Laue patterns using all available neutrons in the white beam. The PCS user facility is described and highlights from the user program are presented.
Acta crystallographica. Section D, Biological crystallography 11/2010; 66(Pt 11):1206-12. · 12.67 Impact Factor
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ABSTRACT: We have investigated the protonation states of histidine residues (potential Bohr groups) in the deoxy form (T state) of human hemoglobin by direct determination of hydrogen (deuterium) positions with the neutron protein crystallography technique. The reversible binding of protons is key to the allosteric regulation of human hemoglobin. The protonation states of 35 of the 38 His residues were directly determined from neutron scattering omit maps, with 3 of the remaining residues being disordered. Protonation states of 5 equivalent His residues--alpha His20, alpha His50, alpha His89, beta His143, and beta His146--differ between the symmetry-related globin subunits. The distal His residues, alpha His58 and beta His63, are protonated in the alpha 1 beta 1 heterodimer and are neutral in alpha 2 beta 2. Buried residue alpha His103 is found to be protonated in both subunits. These distal and buried residues have the potential to act as Bohr groups. The observed protonation states of His residues are compared to changes in their pK(a) values during the transition from the T to the R state and the results provide some new insights into our understanding of the molecular mechanism of the Bohr effect.
Journal of Molecular Biology 03/2010; 398(2):276-91. · 4.00 Impact Factor
