L-Arabinose binding, isomerization, and epimerization by D-xylose isomerase: X-ray/neutron crystallographic and molecular simulation study.

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 (5)S1 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 Ni(2+) cofactors are Michaelis-like and the isomerization between these two sugars proceeds via a cis-ene-diol mechanism.

Using neutron protein crystallography to understand enzyme mechanisms.

A description is given of the results of neutron diffraction studies of the structures of four different metal-ion complexes of deuterated D-xylose isomerase. These represent four stages in the progression of the biochemical catalytic action of this enzyme. Analyses of the structural changes observed between the various three-dimensional structures lead to some insight into the mechanism of action of this enzyme.

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

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.

Hydrogen location in stages of an enzyme-catalyzed reaction: time-of-flight neutron structure of D-xylose isomerase with bound D-xylulose.

The time-of-flight neutron Laue technique has been used to determine the location of hydrogen atoms in the enzyme d-xylose isomerase (XI). The neutron structure of crystalline XI with bound product, d-xylulose, shows, unexpectedly, that O5 of d-xylulose is not protonated but is hydrogen-bonded to doubly protonated His54. Also, Lys289, which is neutral in native XI, is protonated (positively charged), while the catalytic water in native XI has become activated to a hydroxyl anion which is in the proximity of C1 and C2, the molecular site of isomerization of xylose. These findings impact our understanding of the reaction mechanism.

Synthetic, crystallographic, computational, and biological studies of 1,4-difluorobenzo[c]phenanthrene and its metabolites.

1,4-Difluorobenzo[c]phenanthrene (1,4-DFBcPh) and its putative metabolites, the dihydrodiol and diol epoxides, have been synthesized and structurally characterized, and the extent of DNA binding by the metabolites has been assessed. 1,4-DFBcPh and 1,4-difluoro-10-methoxybenzo[c]phenanthrene were prepared by photochemical cyclization of appropriate naphthylphenylethylenes. The dihydrodiol was synthesized from 1,4-difluoro-10-methoxybenzo[c]phenanthrene, and the diol epoxides were diastereoselectively synthesized from the dihydrodiol. Interesting differences were noted in 1H NMR spectra of the series 1 (syn) diol epoxides of benzo[c]phenanthrene (BcPh) and 1,4-DFBcPh; the BcPh diol epoxide displays a quasi-diequatorial orientation of the hydroxyl groups, but in the 1,4-DFBcPh case these are diaxially disposed. This difference probably stems from the presence of the fjord-region fluorine atom in 1,4-DFBcPh. A through-space, fjord-region H-F coupling has also been observed for 1,4-DFBcPh and its derivatives. Comparative X-ray crystallographic analyses of BcPh and 1,4-DFBcPh and their dihydrodiols show that introduction of fluorine increases the molecular distortion by about 6-7 degrees . As a guide to estimating the molecular distortion and its effects, and for comparison with the X-ray structures in known cases, optimized structures of BcPh, 1,4-DFBcPh, and 1,4-DMBcPh (the dimethyl analogue) as well as their dihydrodiols and diol epoxides were computed. Relative aromaticities of these compounds were assessed by nucleus-independent chemical shift calculations, and 13C NMR chemical shifts were computed by gauge-inducing atomic orbital calculations. 1,4-DFBcPh and its dihydrodiol were subjected to metabolism, and the amount of DNA binding in human breast cancer MCF-7 cells was assessed. The extent of DNA binding was then compared with that for BcPh and its dihydrodiol and the potent carcinogen benzo[a]pyrene. The 1,4-DFBcPh series 2 (anti) diol epoxide-derived DNA adducts were also compared with those arising from intracellular oxidation of the dihydrodiol with subsequent DNA binding. These experiments showed that increased molecular distortion decreased metabolic activation to the terminal metabolites but that diol epoxide metabolites that are formed are the DNA-damaging species.

Locating active-site hydrogen atoms in D-xylose isomerase: time-of-flight neutron diffraction.

Time-of-flight neutron diffraction has been used to locate hydrogen atoms that define the ionization states of amino acids in crystals of D-xylose isomerase. This enzyme, from Streptomyces rubiginosus, is one of the largest enzymes studied to date at high resolution (1.8 A) by this method. We have determined the position and orientation of a metal ion-bound water molecule that is located in the active site of the enzyme; this water has been thought to be involved in the isomerization step in which D-xylose is converted to D-xylulose or D-glucose to D-fructose. It is shown to be water (rather than a hydroxyl group) under the conditions of measurement (pH 8.0). Our analyses also reveal that one lysine probably has an -NH(2)-terminal group (rather than NH(3)(+)). The ionization state of each histidine residue also was determined. High-resolution x-ray studies (at 0.94 A) indicate disorder in some side chains when a truncated substrate is bound and suggest how some side chains might move during catalysis. This combination of time-of-flight neutron diffraction and x-ray diffraction can contribute greatly to the elucidation of enzyme mechanisms.

A preliminary time-of-flight neutron diffraction study of Streptomyces rubiginosus D-xylose isomerase.

The metalloenzyme D-xylose isomerase forms well ordered crystals that diffract X-rays to ultrahigh resolution (<1 A). However, structural analysis using X-ray diffraction data has as yet been unable to differentiate between several postulated mechanisms that describe the catalytic activity of this enzyme. Neutrons, with their greater scattering sensitivity to H atoms, could help to resolve this by determining the protonation states within the active site of the enzyme. As the first step in the process of investigating the mechanism of action of D-xylose isomerase from Streptomyces rubiginosus using neutron diffraction, data to better than 2.0 A were measured from the unliganded protein at the Los Alamos Neutron Science Center Protein Crystallography Station. Measurement of these neutron diffraction data represents several milestones: this is one of the largest biological molecules (a tetramer, MW approximately 160 000 Da, with unit-cell lengths around 100 A) ever studied at high resolution using neutron diffraction. It is also one of the first proteins to be studied using time-of-flight techniques. The success of the initial diffraction experiments with D-xylose isomerase demonstrate the power of spallation neutrons for protein crystallography and should provide further impetus for neutron diffraction studies of biologically active and significant proteins. Further data will be measured from the enzyme with bound substrates and inhibitors in order to provide the specific information needed to clarify the catalytic mechanism of this enzyme.

The arrangement of first- and second-shell water molecules in trivalent aluminum complexes: results from density functional theory and structural crystallography.

The structural and energetic features of a variety of gas-phase aluminum ion hydrates containing up to 18 water molecules have been studied computationally using density functional theory. Comparisons are made with experimental data from neutron diffraction studies of aluminum-containing crystal structures listed in the Cambridge Structural Database. Computational studies indicate that the hexahydrated structure Al[H(2)O](6)(3+) (with symmetry T(h)()), in which all six water molecules are located in the innermost coordination shell, is lower in energy than that of Al[H(2)O](5)(3+).[H(2)O], where only five water molecules are in the inner shell and one water molecule is in the second shell. The analogous complex with four water molecules in the inner shell and two in the outer shell undergoes spontaneous proton transfer during the optimization to give [Al[H(2)O](2)[OH](2)](+).[H(3)O(+)](2), which is lower in energy than Al[H(2)O](6)(3+); this finding of H(3)O(+) is consistent with the acidity of concentrated Al(3+) solutions. Since, however, Al[H(2)O](6)(3+) is detected in solutions of Al(3+), additional water molecules are presumed to stabilize the hexa-aquo Al(3+) cation. Three models of a trivalent aluminum ion complex surrounded by a total of 18 water molecules arranged in a first shell containing 6 water molecules and a second shell of 12 water molecules are discussed. We find that a model with S(6) symmetry for which the Al[H(2)O](6)(3+) unit remains essentially octahedral and participates in an integrated hydrogen bonded network with the 12 outer-shell water molecules is lowest in energy. Interactions between the 12 second-shell water molecules and the trivalent aluminum ion in Al[H(2)O](6)(3+) do not appear to be sufficiently strong to orient the dipole moments of these second-shell water molecules toward the Al(3+) ion.

Difluoromethylcobalamin: Structural Aspects of an Old Tree with a New Branch.

Difluoromethylcobalamin (CF(2)Cbl), a vitamin B(12) analogue with CHF(2) replacing CN, can be synthesized in a two-step procedure from aquocobalamin and CHClF(2). Its crystal structure has been determined by X-ray diffraction. The compound crystallizes in the orthorhombic space group P2(1)2(1)2(1) with Z = 4 and 17 water molecules per formula unit. The unit cell dimensions are a = 24.08(1) Å, b = 21.143(3) Å, and c = 15.981(3) Å. The refinement model was kept as simple as possible with no restraints, and with isotropic displacement parameters for all non-hydrogen atoms except for Co, P, and F. The agreement factors obtained this way are: R(1) = 0.072 for 5675 reflections with F(o) > 4 sigma(F(o)) and wR(2) = 0.194 for all 11844 reflections. The packing motif of CF(2)Cbl is very similar to that described for wet vitamin B(12), a distorted hexagonal close packing of the cobalamin, with channels of water running parallel to the crystallographic c axis through the crystal at x = (1)/(4), y = 0, and x = (3)/(4), y = (1)/(2), respectively. An analysis of interactions involving water molecules and amide groups revealed a discontinuity between oxygen-oxygen distances (which are found to be less than 2.8 Å) and oxygen-nitrogen distances (which are found to be greater than 2.8 Å); this provides a useful criterion for distinguishing between oxygen and nitrogen atoms in amide groups. A superposition of the crystal structures of vitamin B(12) and CF(2)Cbl shows a significant change at the molecular level. In CF(2)Cbl, the c side chain of ring B takes on a conformation that brings its terminal amide group near to the CHF(2) group. This results in both a relatively short contact (3.11 Å) between F2 and O39 of the c amide and a weak C1F-H1F.O39 interaction.

Interactions of Metal Ions with Water: Ab Initio Molecular Orbital Studies of Structure, Bonding Enthalpies, Vibrational Frequencies and Charge Distributions. 1. Monohydrates.

The formation and properties of a wide range of metal ion monohydrates, M(n)()(+)-OH(2), where n = 1 and 2, have been studied by ab initio molecular orbital calculations at the MP2(FULL)/6-311++G//MP2(FULL)/6-311++G and CCSD(T)(FULL)/6-311++G//MP2(FULL)/6-311++G computational levels. The ions M are from groups 1A, 2A, 3A, and 4A in the second, third, and fourth periods of the periodic table and the first transition series. Structural parameters, vibrational frequencies, bonding enthalpies, orbital occupancies and energies, and atomic charge distributions are reported. Trends in these properties are correlated with the progressive occupancy of the s, p, and d orbitals. Except for K(+)-OH(2) and Ca(2+)-OH(2), the O-H bond lengths and HOH angles are greater in the hydrates than in unbound water. The M-O bond lengths decrease proceeding from group 1A --> 4A but become larger in proceeding from the second --> fourth period. The bonding enthalpies, are found to be inversely linearly dependent on the M-O bond length M(n)()(+) according to equations of the form = A + B(1/M-O) for n = 1 and n = 2. Within each monohydrate the distribution of atomic charge reveals a small but definite transfer of charge from water to the metal ion. Compared to unbound water there is, in a metal-ion-bound water complex, an increase in the electronic (negative) charge on the oxygen atom, accompanied by a (significantly) larger decrease in the electronic charge on the hydrogen atoms. The bonding of the water molecule, although electrostatic in origin, is thus more complex than a simple interaction between a point charge on the metal ion, and the water dipole.