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• W2086641010 abstract "•Joint X-ray/neutron structures of D-xylose isomerase with L-arabinose•Structures and QM/MM reveal L-arabinose conformation is 5S1 in the active site•MD simulations suggest mutations to improve enzyme affinity to L-arabinose•Isomerization between L-ribulose and L-ribose may proceed by cis-ene-diol pathway D-xylose isomerase (XI) is capable of sugar isomerization and slow conversion of some monosaccharides into their C2-epimers. We present X-ray and neutron crystallographic studies to locate H and D atoms during the respective isomerization and epimerization of L-arabinose to L-ribulose and L-ribose, respectively. Neutron structures in complex with cyclic and linear L-arabinose have demonstrated that the mechanism of ring-opening is the same as for the reaction with D-xylose. Structural evidence and QM/MM calculations show that in the reactive Michaelis complex L-arabinose is distorted to the high-energy 5S1 conformation; this may explain the apparent high KM for this sugar. MD-FEP simulations indicate that amino acid substitutions in a hydrophobic pocket near C5 of L-arabinose can enhance sugar binding. L-ribulose and L-ribose were found in furanose forms when bound to XI. We propose that these complexes containing Ni2+ cofactors are Michaelis-like and the isomerization between these two sugars proceeds via a cis-ene-diol mechanism. D-xylose isomerase (XI) is capable of sugar isomerization and slow conversion of some monosaccharides into their C2-epimers. We present X-ray and neutron crystallographic studies to locate H and D atoms during the respective isomerization and epimerization of L-arabinose to L-ribulose and L-ribose, respectively. Neutron structures in complex with cyclic and linear L-arabinose have demonstrated that the mechanism of ring-opening is the same as for the reaction with D-xylose. Structural evidence and QM/MM calculations show that in the reactive Michaelis complex L-arabinose is distorted to the high-energy 5S1 conformation; this may explain the apparent high KM for this sugar. MD-FEP simulations indicate that amino acid substitutions in a hydrophobic pocket near C5 of L-arabinose can enhance sugar binding. L-ribulose and L-ribose were found in furanose forms when bound to XI. We propose that these complexes containing Ni2+ cofactors are Michaelis-like and the isomerization between these two sugars proceeds via a cis-ene-diol mechanism. The bacterial and fungal metalloenzyme D-xylose isomerase (XI, EC 5.3.1.5), first discovered in Aerobactor cloacae (Tsumura and Sato, 1961Tsumura N. Sato T. Enzymatic conversion of D-glucose to D-fructose. I. Identification of active bacterial strain and confirmation of D-fructose formation.Agric. Biol. Chem. 1961; 25: 616-619Crossref Scopus (4) Google Scholar), catalyzes the reversible aldose-ketose isomerization of both D- and L-enantiomers of several hexose and pentose monomeric sugars (Figure 1). Intensive research efforts are underway to improve the suitability of XI for industrial applications that include the conversion of D-xylose to D-xylulose during the production of biofuels from lignocellulosic biomass (van Maris et al., 2006van Maris A.J.A. Abbott D.A. Bellissimi E. van den Brink J. Kuyper M. Luttik M.A.H. Wisselink H.W. Scheffers W.A. van Dijken J.P. Pronk J.T. Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status.Antonie van Leeuwenhoek. 2006; 90: 391-418Crossref PubMed Scopus (394) Google Scholar, van Maris et al., 2007van Maris A.J.A. Winkler A.A. Kuyper M. de Laat W.T.A.M. van Dijken J.P. Pronk J.T. Development of efficient xylose fermentation in Saccharomyces cerevisiae: xylose isomerase as a key component.Adv. Biochem. Eng. Biotechnol. 2007; 108: 179-204PubMed Google Scholar), the conversion of D-glucose to D-fructose during the production of starch-based high-fructose corn syrup (Bhosale et al., 1996Bhosale S.H. Rao M.B. Deshpande V.V. Molecular and industrial aspects of glucose isomerase.Microbiol. Rev. 1996; 60: 280-300Crossref PubMed Google Scholar, Hartley et al., 2000Hartley B.S. Hanlon N. Jackson R.J. Rangarajan M. Glucose isomerase: insights into protein engineering for increased thermostability.Biochim. Biophys. Acta. 2000; 1543: 294-335Crossref PubMed Scopus (76) Google Scholar), and perhaps ironically, the treatment of human gastrointestinal problems due to fructose malabsorption (Komericki et al., 2012Komericki P. Akkilic-Materna M. Strimitzer T. Weyermair K. Hammer H.F. Aberer W. Oral xylose isomerase decreases breath hydrogen excretion and improves gastrointestinal symptoms in fructose malabsorption - a double-blind, placebo-controlled study.Aliment. Pharmacol. Ther. 2012; 36: 980-987Crossref PubMed Scopus (20) Google Scholar). In addition to isomerization, it has been shown that XI catalyzes the epimerization of the D- and L-forms of xylose and arabinose, thereby producing the C-2 epimers lyxose and ribose, respectively (Figure 1), and this has attracted further industrial attention (Pastinen et al., 1999aPastinen O. Schoemaker H.E. Leisola M. Xylose isomerase catalyzed novel hexose epimerization.Biocatalysis Biotransform. 1999; 17: 393-400Crossref Scopus (20) Google Scholar, Pastinen et al., 1999bPastinen O. Visuri K. Schoemaker H.E. Leisola M. Novel reactions of xylose isomerase from Streptomyces rubiginosus.Enzyme Microb. Technol. 1999; 25: 695-700Crossref Scopus (28) Google Scholar); L-ribose in particular, an unnatural enantiomer of D-ribose, has important commercial and biomedical potential as a precursor for the synthesis of different antiviral and anticancer drugs (Okano, 2009Okano K. Synthesis and pharmaceutical application of L-ribose.Tetrahedron. 2009; 65: 1937-1949Crossref Scopus (51) Google Scholar). L-ribose can be synthesized from L-arabinose by multistep chemical epimerization using transition metal catalysts (Zemek et al., 1975Zemek J. Bílik V. Zákutná L. Effect of some aldoses on growth of Saccharomyces cerevisiae inhibited with molybdenum.Folia Microbiol. (Praha). 1975; 20: 467-469Crossref PubMed Scopus (3) Google Scholar, Akagi et al., 2002Akagi M. Omae D. Tamura Y. Ueda T. Kumashiro T. Urata H. A practical synthesis of L-ribose.Chem. Pharm. Bull. (Tokyo). 2002; 50: 866-868Crossref PubMed Scopus (29) Google Scholar, Jeon et al., 2010Jeon Y.-J. Park M.B. Kim I.-H. L-Ribose from L-arabinose by epimerization and its purification by 3-zone simulated moving bed chromatography.Bioprocess Biosyst. Eng. 2010; 33: 87-95Crossref PubMed Scopus (17) Google Scholar) or from other sugars by a series of chemical reactions (Yun et al., 2005Yun M. Moon H.R. Kim H.O. Choi W.J. Kim Y.C. Park C.S. Jeong L.S. A highly efficient synthesis of unnatural L-sugars from D-ribose.Tetrahedron Lett. 2005; 46: 5903-5905Crossref Scopus (54) Google Scholar, Perali et al., 2011Perali R.S. Mandava S. Bandi R. A convenient synthesis of L-ribose from D-fructose.Tetrahedron. 2011; 67: 4031-4035Crossref Scopus (26) Google Scholar), but these approaches require purified substrates that reduce cost-effectiveness. An advantage of alternative biotechnological approaches is that enzymes can work effectively on crude biomass pulps or hydrolysates, e.g., beet pulp, which contains ∼20% L-arabinose (Helanto et al., 2009Helanto M. Kiviharju K. Granström T. Leisola M. Nyyssölä A. Biotechnological production of L-ribose from L-arabinose.Appl. Microbiol. Biotechnol. 2009; 83: 77-83Crossref PubMed Scopus (41) Google Scholar, Hu et al., 2011Hu C. Li L. Zheng Y. Rui L. Hu C. Perspectives of biotechnological production of L-ribose and its purification.Appl. Microbiol. Biotechnol. 2011; 92: 449-455Crossref PubMed Scopus (32) Google Scholar). Crystalline cross-linked XI from Streptomyces rubiginosus (Leisola et al., 2001Leisola M. Jokela J. Finell J. Pastinen O. Simultaneous catalysis and product separation by cross-linked enzyme crystals.Biotechnol. Bioeng. 2001; 72: 501-505Crossref PubMed Scopus (22) Google Scholar, Jokela et al., 2002Jokela J. Pastinen O. Leisola M. Isomerization of pentose and hexose sugars by an enzyme reactor packed with cross-linked xylose isomerase crystals.Enzyme Microb. Technol. 2002; 31: 67-76Crossref Scopus (48) Google Scholar) has been commercialized to produce L-ribose from L-arabinose. However, the epimerization reaction is slow, requires elevated temperatures, and the equilibrium mixture contains ∼53% L-arabinose, 22% L-ribulose, and 25% L-ribose. One approach to improving the suitability of XI for commercial production of L-ribose is protein engineering. However, high-throughput engineering using directed evolution has had limited success so far in improving the isomerization reaction for XI (Sriprapundh et al., 2003Sriprapundh D. Vieille C. Zeikus J.G. Directed evolution of Thermotoga neapolitana xylose isomerase: high activity on glucose at low temperature and low pH.Protein Eng. 2003; 16: 683-690Crossref PubMed Scopus (64) Google Scholar, Lee et al., 2012Lee S.-M. Jellison T. Alper H.S. Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae.Appl. Environ. Microbiol. 2012; 78: 5708-5716Crossref PubMed Scopus (115) Google Scholar, Zhou et al., 2012Zhou H. Cheng J.S. Wang B.L. Fink G.R. Stephanopoulos G. Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae.Metab. Eng. 2012; 14: 611-622Crossref PubMed Scopus (219) Google Scholar). An alternative or complementary approach involves rational engineering. Informed site-directed mutagenesis requires an understanding of the catalytic mechanisms involved, but the complex multistep mechanisms of both isomerization and epimerization by XI are not yet adequately understood for this purpose. The structures of XI from various species have been characterized with X-ray crystallography (Carrell et al., 1984Carrell H.L. Rubin B.H. Hurley T.J. Glusker J.P. X-ray crystal structure of D-xylose isomerase at 4-A resolution.J. Biol. Chem. 1984; 259: 3230-3236PubMed Google Scholar, Carrell et al., 1989Carrell H.L. Glusker J.P. Burger V. Manfre F. Tritsch D. Biellmann J.-F. X-ray analysis of D-xylose isomerase at 1.9 A: native enzyme in complex with substrate and with a mechanism-designed inactivator.Proc. Natl. Acad. Sci. USA. 1989; 86: 4440-4444Crossref PubMed Scopus (197) Google Scholar, Collyer et al., 1990Collyer C.A. Henrick K. Blow D.M. Mechanism for aldose-ketose interconversion by D-xylose isomerase involving ring opening followed by a 1,2-hydride shift.J. Mol. Biol. 1990; 212: 211-235Crossref PubMed Scopus (154) Google Scholar, Allen et al., 1994Allen K.N. Lavie A. Glasfeld A. Tanada T.N. Gerrity D.P. Carlson S.C. Farber G.K. Petsko G.A. Ringe D. Role of the divalent metal ion in sugar binding, ring opening, and isomerization by D-xylose isomerase: replacement of a catalytic metal by an amino acid.Biochemistry. 1994; 33: 1488-1494Crossref PubMed Scopus (66) Google Scholar, Fuxreiter et al., 1997Fuxreiter M. Böcskei Z. Szeibert A. Szabó E. Dallmann G. Naray-Szabo G. Asboth B. Role of electrostatics at the catalytic metal binding site in xylose isomerase action: Ca(2+)-inhibition and metal competence in the double mutant D254E/D256E.Proteins. 1997; 28: 183-193Crossref PubMed Scopus (20) Google Scholar, Fenn et al., 2004Fenn T.D. Ringe D. Petsko G.A. Xylose isomerase in substrate and inhibitor Michaelis states: atomic resolution studies of a metal-mediated hydride shift.Biochemistry. 2004; 43: 6464-6474Crossref PubMed Scopus (95) Google Scholar). The mechanism of D-xylose to D-xylulose (and D-glucose to D-fructose) isomerization has been examined extensively using both experiment and theory (Asbóth and Náray-Szabó, 2000Asbóth B. Náray-Szabó G. Mechanism of action of D-xylose isomerase.Curr. Protein Pept. Sci. 2000; 1: 237-254Crossref PubMed Scopus (55) Google Scholar, Garcia-Viloca et al., 2002Garcia-Viloca M. Alhambra C. Truhlar D.G. Gao J. Quantum dynamics of hydride transfer catalyzed by bimetallic electrophilic catalysis: synchronous motion of Mg2+ and H− in xylose isomerase.J. Am. Chem. Soc. 2002; 124: 7268-7269Crossref PubMed Scopus (32) Google Scholar, Garcia-Viloca et al., 2003Garcia-Viloca M. Alhambra C. Truhlar D.G. Gao J. Hydride transfer catalyzed by xylose isomerase: mechanism and quantum effects.J. Comput. Chem. 2003; 24: 177-190Crossref PubMed Scopus (88) Google Scholar). XI from Streptomyces rubiginosus, the most studied example, is a tetramer of identical subunits (molecular weight 172 kDa), each with a (βα)8 barrel fold and an active site located at the C terminus of the β-barrel. Magnesium is the physiological metal found at two binding sites in the shoebox-shaped active site, M1 and M2, binding directly to water molecules as well as to active-site residues. M1 is referred to as the structural metal, whereas M2 is called the catalytic metal (Whitlow et al., 1991Whitlow M. Howard A.J. Finzel B.C. Poulos T.L. Winborne E. Gilliland G.L. A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and D-xylose.Proteins. 1991; 9: 153-173Crossref PubMed Scopus (187) Google Scholar). M1 binds Glu181, Glu217, Asp245, Asp287, and two water molecules (W2 and W3); M2 binds Glu217 (shared with M1), His220, Asp255 (bidentate), Asp257, and the catalytic water (see Figure 2 in Kovalevsky et al., 2010Kovalevsky A.Y. Hanson L. Fisher S.Z. Mustyakimov M. Mason S.A. Forsyth V.T. Blakeley M.P. Keen D.A. Wagner T. Carrell H.L. et al.Metal ion roles and the movement of hydrogen during reaction catalyzed by D-xylose isomerase: a joint x-ray and neutron diffraction study.Structure. 2010; 18: 688-699Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Several mechanisms of isomerization consistent with X-ray structures have been proposed which differ in the exact location and mode of transfer of H at each step (Collyer et al., 1990Collyer C.A. Henrick K. Blow D.M. Mechanism for aldose-ketose interconversion by D-xylose isomerase involving ring opening followed by a 1,2-hydride shift.J. Mol. Biol. 1990; 212: 211-235Crossref PubMed Scopus (154) Google Scholar, Fenn et al., 2004Fenn T.D. Ringe D. Petsko G.A. Xylose isomerase in substrate and inhibitor Michaelis states: atomic resolution studies of a metal-mediated hydride shift.Biochemistry. 2004; 43: 6464-6474Crossref PubMed Scopus (95) Google Scholar, Allen et al., 1994Allen K.N. Lavie A. Glasfeld A. Tanada T.N. Gerrity D.P. Carlson S.C. Farber G.K. Petsko G.A. Ringe D. Role of the divalent metal ion in sugar binding, ring opening, and isomerization by D-xylose isomerase: replacement of a catalytic metal by an amino acid.Biochemistry. 1994; 33: 1488-1494Crossref PubMed Scopus (66) Google Scholar, Bogumil et al., 1997Bogumil R. Kappl R. Hüttermann J. Witzel H. Electron paramagnetic resonance of D-xylose isomerase: evidence for metal ion movement induced by binding of cyclic substrates and inhibitors.Biochemistry. 1997; 36: 2345-2352Crossref PubMed Scopus (21) Google Scholar). A limitation of X-ray studies is the inability to easily determine the location of H atoms in XI, and therefore to differentiate between these proposals. To overcome this limitation, we therefore applied neutron crystallography, as neutrons can provide H locations, especially if H is replaced by its isotope, deuterium (D). Exchangeable H (attached to N, O or S) can be replaced by D by soaking the enzyme in D2O. The neutron work has led to new suggestions as to how structural changes might take place over the course of the reaction. In particular, we previously identified key residues involved in pH-dependent metal cofactor binding, the catalytic steps of ring opening of the cyclic substrate, and isomerization of the linear intermediate (Katz et al., 2006Katz A.K. Li X. Carrell H.L. Hanson B.L. Langan P. Coates L. Schoenborn B.P. Glusker J.P. Bunick G.J. Locating active-site hydrogen atoms in D-xylose isomerase: time-of-flight neutron diffraction.Proc. Natl. Acad. Sci. USA. 2006; 103: 8342-8347Crossref PubMed Scopus (59) Google Scholar, Kovalevsky et al., 2008Kovalevsky A.Y. Katz A.K. Carrell H.L. Hanson L. Mustyakimov M. Fisher S.Z. Coates L. Schoenborn B.P. Bunick G.J. Glusker J.P. Langan P. Hydrogen location in stages of an enzyme-catalyzed reaction: time-of-flight neutron structure of D-xylose isomerase with bound D-xylulose.Biochemistry. 2008; 47: 7595-7597Crossref PubMed Scopus (40) Google Scholar, Kovalevsky et al., 2010Kovalevsky A.Y. Hanson L. Fisher S.Z. Mustyakimov M. Mason S.A. Forsyth V.T. Blakeley M.P. Keen D.A. Wagner T. Carrell H.L. et al.Metal ion roles and the movement of hydrogen during reaction catalyzed by D-xylose isomerase: a joint x-ray and neutron diffraction study.Structure. 2010; 18: 688-699Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, Kovalevsky et al., 2011Kovalevsky A.Y. Hanson B.L. Mason S.A. Yoshida T. Fisher S.Z. Mustyakimov M. Forsyth V.T. Blakeley M.P. Keen D.A. Langan P. Identification of the elusive hydronium ion exchanging roles with a proton in an enzyme at lower pH values.Angew. Chem. Int. Ed. Engl. 2011; 50: 7520-7523Crossref PubMed Scopus (57) Google Scholar). We then used this information to guide site-directed mutagenesis in rational protein engineering studies, which significantly improved the XI activity (kcat/KM) for D-xylose isomerization at low pH (Waltman et al., 2014Waltman M.J. Yang Z.K. Langan P. Graham D.E. Kovalevsky A. Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design.Protein Eng. Des. Sel. 2014; 27: 59-64Crossref PubMed Scopus (14) Google Scholar). Here, we report further X-ray and neutron (XN) crystallographic studies of XI, this time with the aim of understanding the catalytic mechanism of isomerization of L-arabinose to L-ribulose and the epimerization of L-ribulose to L-ribose, for which no reliable mechanistic information is available. Further, we augment this crystallographic approach with quantum mechanical/molecular mechanical (QM/MM) calculations and molecular dynamics (MD) simulations, which help interpret the experimental findings and predict favorable mutations for rational protein engineering. We report two neutron structures of ternary complexes containing L-arabinose and either Cd2+ or Ni2+, designated XI-Cd-CyclicArabinose_n and XI-Ni-LinearArabinose_n. As with D-glucose (Kovalevsky et al., 2010Kovalevsky A.Y. Hanson L. Fisher S.Z. Mustyakimov M. Mason S.A. Forsyth V.T. Blakeley M.P. Keen D.A. Wagner T. Carrell H.L. et al.Metal ion roles and the movement of hydrogen during reaction catalyzed by D-xylose isomerase: a joint x-ray and neutron diffraction study.Structure. 2010; 18: 688-699Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), Cd2+ prevents ring opening, resulting in a Michaelis-like complex containing cyclic L-arabinose. Then Ni2+ inhibits XI at the next step after the ring has been opened but before isomerization occurs, with linear L-arabinose observed in the active site. We find that the active sites in XI-Cd-CyclicArabinose_n and XI-Ni-LinearArabinose_n are similar to those in neutron structures with cyclic and linear D-glucose (Kovalevsky et al., 2010Kovalevsky A.Y. Hanson L. Fisher S.Z. Mustyakimov M. Mason S.A. Forsyth V.T. Blakeley M.P. Keen D.A. Wagner T. Carrell H.L. et al.Metal ion roles and the movement of hydrogen during reaction catalyzed by D-xylose isomerase: a joint x-ray and neutron diffraction study.Structure. 2010; 18: 688-699Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), indicating that the mechanism of ring opening and isomerization for L-arabinose is similar to that for D-xylose and D-glucose. Unexpectedly, we find that in XI-Cd-CyclicArabinose_n the β-L-arabinose anomer adopts a high-energy 5S1 conformation instead of the low-energy 4C1 conformation of D-xylose and D-glucose observed in previously reported XI Michaelis-like complexes, providing insight into the very weak apparent Michaelis constant (KM) for L-arabinose (Karimäki et al., 2004Karimäki J. Parkkinen T. Santa H. Pastinen O. Leisola M. Rouvinen J. Turunen O. Engineering the substrate specificity of xylose isomerase.Protein Eng. Des. Sel. 2004; 17: 861-869Crossref PubMed Scopus (33) Google Scholar, Patel et al., 2012Patel D.H. Cho E.J. Kim H.M. Choi I.S. Bae H.-J. Engineering of the catalytic site of xylose isomerase to enhance bioconversion of a non-preferential substrate.Protein Eng. Des. Sel. 2012; 25: 331-336Crossref PubMed Scopus (7) Google Scholar). QM/MM calculations based on XI-Cd-CyclicArabinose_n and the physiologically relevant XI-Mg-CyclicArabinose support the experimental observation of the high-energy 5S1 conformation, models containing the 5S1 conformation for L-arabinose being favored over those containing the 4C1 conformation by 10–11 kcal mol−1. In silico site-directed mutagenesis and MD free-energy perturbation (FEP) simulations performed on the XI-Mg-CyclicArabinose model suggest substitutions in the vicinity of L-arabinose (specifically, Thr90, Val135, and Met88) that may result in improved binding affinity for the 5S1 conformation of the sugar. The simulations also reveal the importance of Thr90 in making a water-mediated interaction with a substrate and the significance of conformational freedom of the Met88 side chain. We have also determined X-ray structures of XI in complex with L-ribulose and L-ribose and either Cd2+, Ni2+ or Mg2+ to help understand the epimerization of L-arabinose to L-ribose. These structures are designated XI-M-Ribulose_x and XI-M-Ribose_x in the text, where M is Cd, Ni, or Mg. Again, surprisingly, L-ribulose and L-ribose bind to XI in completely different orientations than cyclic D-glucose, D-xylose, or L-arabinose, and there is no evidence of ring opening even with physiological Mg2+. Additionally, L-ribose is found in the active site as ribofuranose instead of ribopyranose, the latter being the main sugar form in solution and probably also the reactive species for XI. These unexpected sugar orientations would not easily allow ring opening and metal-assisted isomerization using the same hydride shift mechanism suggested for D-glucose, D-xylose, and L-arabinose. One interpretation of this result is that the sugars have bound to XI nonproductively and therefore inhibit the reaction. However, we also discuss the possibility that the structures represent the Michaelis-like complexes of a different XI catalytic mechanism of epimerization. Key findings of the current study are: (1) the non-physiological substrate L-arabinose binds to the XI active site in the high-energy 5S1 skew-boat conformation; (2) QM/MM calculations provide evidence that the XI complex containing the 5S1 conformation of L-arabinose is 10–11 kcal/mol more stable than that with the low-energy 4C1 conformation; (3) the isomerization of L-arabinose to L-ribulose is likely to occur through a similar mechanism as was proposed for the conversion of D-xylose into D-xylulose; (4) molecular dynamics simulations suggest possible amino acid substitutions in the sugar binding site that may improve the XI affinity for L-arabinose; (5) L-ribulose and L-ribose bind to XI as furanoses, having α- or β-orientation of the hydroxyl at the anomeric carbon depending on the metal species present; and (6) the isomerization between L-ribulose and L-ribose may proceed through a cis-ene-diol mechanism involving Glu181 and Asp287. The neutron data obtained for the two XI complexes with L-arabinose are summarized in Table 1, and the X-ray data for the five XI complexes with L-ribulose and L-ribose are provided in Table S1 (available online).Table 1Room Temperature Neutron Crystallographic Data Collection and Joint XN Refinement StatisticsParameterXI-Cd-CyclicArabinose_n PDB ID 4QDPXI-Ni-LinearArabinose_n PDB ID 4QDWData collectionBeamline/facilityPCS/LANSCED19/ILLSpace groupI222I222Cell dimensions a, b, c (Å)93.93, 99.69, 102.9794.19, 99.67, 102.94 α, β, γ (°)90, 90, 9090, 90, 90Resolution (Å)39.28–2.00 (2.11–2.00)aValues in parentheses are for highest-resolution shell. Data were collected from 1 crystal for each structure.71.61–1.80 (1.90–1.80)No. reflections measured101,968 (6,472)101,727 (10,439)No. reflections unique28,786 (3,459)36,814 (5,461)Rmerge0.208 (0.370)0.120 (0.490)I/σI5.5 (1.6)10.1 (1.5)Completeness (%)88.3 (74.0)81.5 (81.6)Redundancy3.5 (1.9)2.7 (1.9)Data rejection criteriano observation & |F| = 0no observation & |F| = 0Joint XN refinementResolution (neutron, Å)20–2.0020–1.80Resolution (X-ray, Å)20–1.6020–1.60Sigma cut-off2.52.0No. reflections (neutron)25,24528,443No. reflections (X-ray)60,03859,926Rwork/Rfree (neutron)0.231 / 0.2470.166 / 0.179Rwork/Rfree (X-ray)0.172 / 0.1850.181/ 0.190No. atomsProtein including H and D5,9845,983Metal22Sugar2019Water936 (312 D2O's)777 (259 D2O's)B-factorsProtein28.424.9Sugar/metal33.032.7Water40.543.2RmsdsBond lengths (Å)0.0070.005Bond angles (°)0.9960.914a Values in parentheses are for highest-resolution shell. Data were collected from 1 crystal for each structure. Open table in a new tab In XI-Cd-CyclicArabinose_n, the cyclic pyranose form of H/D-exchanged L-arabinose is observed, as evidenced by the omit difference FO-FC electron density map shown in Figure 2A. As expected, only the hydroxyl groups of the sugar underwent H/D exchange to OD groups, whereas CH groups remained unchanged. L-arabinopyranose coordinates to M1 with O3 and O4, whereas the endocyclic O5 is hydrogen bonded to His54 on the other side of the sugar. The six-membered pyranose ring of L-arabinose adopts a 5S1 conformation, which is different from the usual 4C1 conformation of D-glucose observed in XI-Cd-CyclicGlucose_n and earlier XI X-ray structures (Carrell et al., 1994Carrell H.L. Hoier H. Glusker J.P. Modes of binding substrates and their analogues to the enzyme D-xylose isomerase.Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 113-123Crossref PubMed Scopus (55) Google Scholar, Fenn et al., 2004Fenn T.D. Ringe D. Petsko G.A. Xylose isomerase in substrate and inhibitor Michaelis states: atomic resolution studies of a metal-mediated hydride shift.Biochemistry. 2004; 43: 6464-6474Crossref PubMed Scopus (95) Google Scholar, Kovalevsky et al., 2010Kovalevsky A.Y. Hanson L. Fisher S.Z. Mustyakimov M. Mason S.A. Forsyth V.T. Blakeley M.P. Keen D.A. Wagner T. Carrell H.L. et al.Metal ion roles and the movement of hydrogen during reaction catalyzed by D-xylose isomerase: a joint x-ray and neutron diffraction study.Structure. 2010; 18: 688-699Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). O4 of L-arabinopyranose has the opposite stereochemistry to that in D-glucose (and D-xylose). As such, in the 4C1 conformation the O4 of L-arabinopyranose is in the axial position, rather than the equatorial position in D-glucose, which would preclude the formation of a coordination bond to M1 (see detailed comparison of 5S1 and 4C1 L-arabinose conformers binding to XI below). Moreover, it is the β-anomer of L-arabinopyranose that is observed in the active site. The neutron density map of the active site is depicted in Figure 2B. O4 donates its D in a hydrogen bond with the carboxylate side chain of Glu181, with an O···O separation of 2.5 Å. Such a short contact is also present for D-glucose in XI-Cd-CyclicGlucose_n. O3 forms a 3.1 Å interaction with the catalytic water molecule, which is significantly longer than the similar contact of 2.7 Å made by D-glucose in XI-Cd-CyclicGlucose_n. O5 of L-arabinose accepts a D from Nε2 of His54 in a 2.7 Å hydrogen bond and makes a weaker water-mediated interaction with Thr90. There are many similarities between the active sites with bound cyclic pentose (XI-Cd-CyclicArabinose_n) and hexose (XI-Cd-CyclicGlucose_n) sugars (Figure 2C), but also some differences, especially in the hydration structure. L-arabinose and D-glucose occupy very similar positions on binding to XI. L-arabinose lacks a bulky hydroxymethylene substituent at C5, which in XI-Cd-CyclicGlucose_n is located in the hydrophobic pocket lined by Met88, Thr90, and Val135, making no hydrogen bond interactions. In XI-Cd-CyclicArabinose_n no extra water molecules enter this pocket and it remains empty. D2Ocat and W1, W2, W3, W4, W6, and W7 remain in the active site, but change their orientations, except for D2Ocat. W1 accepts a D from Lys183 in a hydrogen bond in XI-Cd-CyclicGlucose_n and acts as a hydrogen bond donor to O2 of D-glucose. In XI-Cd-CyclicArabinose_n, W1 flips 180° accompanied by a similar rotation of the O2-D group of the sugar, accepting D atoms in hydrogen bonds with ND3+ of Lys183 and O2. In addition, W2 rotates toward O5 of L-arabinose to form a weak hydrogen bond, whereas it faces away from D-glucose toward the main-chain carbonyl of Thr90. In XI-Cd-CyclicArabinose_n, the O1 hydroxyl of L-arabinose is in the axial position, occupying the same position as it does in D-glucose, and has a similar O-D orientation in both sugars. In XI-Cd-CyclicGlucose_n, O1-D is hydrogen bonded to W7, which in turn is connected to the neutral amine (ND2) of Lys289 through two more waters. This network of water molecules connecting O1 of the sugar and Lys289 is altered in XI-Cd-CyclicArabinose_n, where this connection is accomplished through W4 and W5. W4 remains in both structures; however, W5 is absent when D-glucose is bound. Perhaps this change in the locations of water molecules between the active sites of XI-Cd-CyclicGlucose_n and XI-Cd-CyclicArabinose_n results in slightly different conformations of the Lys289 side chains. The terminal ND2 group of Lys289 is rotated toward Asp257 in the former structure and away from Asp257 in the latter structure, while the aliphatic chains and Nζ atoms maintain thei" @default.
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• W2086641010 title "L-Arabinose Binding, Isomerization, and Epimerization by D-Xylose Isomerase: X-Ray/Neutron Crystallographic and Molecular Simulation Study" @default.
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