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.

Unusually long cooperative chain of seven hydrogen bonds. An alternative packing type for symmetrical phenols.

Conformational flexibility in a symmetrical tris-phenol leads to close packed structures that are also characterised by an extended though finite cooperative chain of hydrogen bonds.

Three-dimensional structure of anti-5,6-dimethylchrysene-1, 2-dihydrodiol-3,4-epoxide: a diol epoxide with a bay region methyl group.

The three-dimensional structure of a dihydrodiol epoxide of 5, 6-dimethylchrysene was elucidated by X-ray diffraction techniques. The effects of the steric overcrowding by the 5-methyl group in the bay region of this compound are described. The carbon atom of the 5-methyl group is found to lie out of the plane of the aromatic system, thereby avoiding the nearer C-H group of the epoxide ring; this C-H hydrogen atom is pushed in the opposite direction. As a result, the molecule is distorted so that the relative orientations of the epoxide group and the aromatic ring systems are very different for the diol epoxides of (nearly planar) benzo[a]pyrene (studied by Neidle and co-workers) and (distorted) 5, 6-dimethylchrysene (described here). The main effect of the 5-methyl group is to change the relative angle between the epoxide-bearing ring (the site of attack when the diol epoxide acts as an alkylating agent) and the aromatic ring system (which is presumed to lie partially between the DNA bases in the DNA adduct that is about to be formed). This may favor some specific alkylation geometry.

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.

Crystallization and preliminary X-ray diffraction studies of E. coli porphobilinogen synthase and its heavy-atom derivatives.

Porphobilinogen synthase (PBGS) catalyzes the condensation of two identical substrate molecules, 5-aminolevulinic acid (ALA), in an asymmetric manner to form porphobilinogen. E. coli PBGS is an homooctameric enzyme. The number of active sites is not clear, but each subunit binds one ZnII ion and one MgII ion. Diffraction-quality crystals of native E. coli PBGS have been obtained, and unit-cell dimensions (a = 130.8, c = 144.0 A) are reported. These crystals diffract to about 3.0 A resolution.

Dibenzo[a,l]pyrene (dibenzo[def,p]chrysene): fjord-region distortions.

The molecular dimensions of the potent chemical carcinogen dibenzo[def,p]chrysene, also known as dibenzo[a,l]pyrene, have been determined by X-ray diffraction methods. This analysis shows that the molecule is considerably distorted so that it is non-planar with an angle of 27.6 degrees between the outermost rings and a widening of C-C-C bond angles in the fjord region. The dimensions of the molecular distortion due to atomic overcrowding in the fjord region are presented. This polycyclic aromatic hydrocarbon is a more potent carcinogen than is benzo[a]pyrene or its 11-methyl derivative. Comparisons of the distortions in dibenzo[a,l]pyrene with the geometries of various other polycyclic aromatic hydrocarbons containing fjord- or bay-region methyl groups provide structural data on the ratio of angular to torsional distortion in such overcrowded molecules.

Trimethyl isocyanurate and triethyl isocyanurate.

The crystal structures of trimethyl isocyanurate, C6H9N3O3, (1), and triethyl isocyanurate, C9H15N3O3, (2), contain topologically similar C--H...O hydrogen-bonded networks. In (1), there are two symmetry-independent molecules and each forms its own layer structure. In (2), two of the ethyl groups point one way with respect to the heterocyclic ring, while the third points in the opposite direction.

Benzo[a]pyrene and its analogues: structural studies of molecular strain.

The molecular geometry of benzo[a]pyrene, its 4-methyl-and 3,11-dimethyl derivatives, benzo[e]pyrene, and two azabenzo[a]pyrenes are described. Results of these three-dimensional crystal structure determinations, together with those from previous studies in this laboratory of 11-methylbenzo[a]pyrene, indicate the extent to which nonbonded interactions between hydrogen atoms contribute to molecular distortions, particularly in the bay-region. This strain is high if a bay-region methyl group is present. The major effect is an increase in the C-C-C angles in that area of the molecule, rather than torsion about bonds. In addition, the effect of a nitrogen atom replacing one of the C-H groups in the aromatic system is shown. Molecules stack in planes approximately 3.5 A apart. In benzo[a]pyrene, 5-azabenzo[a]pyrene and 3,11-dimethylbenzo[a]pyrene crystals the stacking is similar to that in graphite. 4-Methylbenzo[a]pyrene molecules stack with less molecular overlap. The packing in 4-aza-5-methylbenzo[a]pyrene consists of modules of four stacked molecules, packed in a 'tile-like' arrangement. Nonbonded C....H interactions between adjacent molecules lead to a herring-bone arrangement between these stacks. The types of C....H and pi-pi interactions involving PAHs in the crystalline state, described here, can also be expected to be found when the PAHs bind to hydrophobic areas of biological macromolecules such as proteins, nucleic acids and membranes.

Bay-region distortions in a methanol adduct of a bay-region diol epoxide of the carcinogen 5-methylchrysene.

The three-dimensional structure of the product of the reaction of a diol epoxide of the carcinogen 5-methylchrysene with methanol has been determined by an X-ray diffraction analysis. The diol epoxide used to obtain this compound contains a stereochemically hindered bay region because of the location of the 5-methyl group, and this might be expected to affect the type of chemical reaction that occurs. The crystal structure analysis of this adduct of a polycyclic aromatic hydrocarbon (PAH) showed that a methoxy group has been added at the carbon atom of the epoxy group that is nearest to the aromatic system. The bond that is formed is axial to the ring system so that the carbon and hydrogen atoms of the methoxy group are considerably displaced from the PAH ring plane. The bay-region methyl group at position 5 is displaced out of the ring plane in the opposite direction. The major steric distortion in this methanol adduct is shown, by a comparison with crystal structures of related non-methylated compounds, to be in the area of the 5-methyl group and not in the tetrol-bearing ring. The steric effects that caused the axial conformation of the newly formed bond would also be expected to pertain in the DNA adduct of a PAH with a bay-region methyl group. Since the presence of the bay-region methyl group in 5-methylchrysene has been shown to enhance the carcinogenicity of this PAH over the parent compound or compounds with methyl groups in other positions of the molecule, it might be anticipated that this axial bond is found in carcinogenic lesions in DNA, and that any factor that ensures this axial conformation may accentuate the carcinogenic potential of a PAH of the appropriate size.

The structure of a coumarin derivative related to the carcinogen benz[a]anthracene.

The three-dimensional structure of 3-methyl-2H-anthra[1,2-b]pyran-2-one, an anticarcinogenic coumarin related to the carcinogen benz[a]anthracene, has been determined by X-ray diffraction techniques. The molecule, apart from hydrogen atoms in the methyl group, is flat, the maximum deviation from its least squares best plane being 0.13 angstroms. The carbonyl C=O bond length is normal [1.206(1) angstroms] and the bonding throughout the molecule indicates localization of double bonds within the coumarin ring, but some delocalization of electrons in the other rings. Molecules pack in planes parallel to each other, the coumarin ring oxygen atom lying between two aromatic rings of other coumarin molecules. The bulky methyl groups are not involved in such stacking, while the carbonyl groups attract C-H groups in neighboring molecules by way of C-H...O interactions. These are the types of interactions that such coumarins could make if they bound to hydrophobic areas in biological macromolecules.

Crystallization and preliminary X-ray diffraction studies of 5-chlorolevulinate-modified bovine porphobilinogen synthase and the Pb(II)-complexed enzyme.

Bovine porphobilinogen synthase (PBGS) is an homo-octameric enzyme with four active sites. Each active site binds two Zn(II) atoms whose ligands differ and two molecules of 5-aminolevulinate whose chemical fates differ. The asymmetric binding of two Zn(II) atoms and two identical substrate molecules by a homodimeric active site is apparently unique. Modification by 5-chiorolevulinate can be used to differentiate the two substrate-binding sites; diffraction-quality crystals of 5-chlorolevulinate-modified PBGS have been obtained. Pb(II) can be used to differentiate the two different Zn(II)-binding sites; diffraction-quality crystals of the Pb(II) complex of PBGS have been obtained. Preliminary diffraction data reveal an I422 space group, in agreement with a general model for the quaternary structure of PBGS.

2-bromoacetoxybenzoic acid, a brominated aspirin analog.

The crystal structure of 2-bromoacetoxybenzoic acid, C9H7BrO4, shows it to be a close structural analog of aspirin. The carboxylic acid moiety is twisted by 7.7 (4) degrees out of the plane of the aromatic ring. The acetyl group, like that of aspirin, shows bond-angle distortions from ideal values while remaining essentially planar. The Br atom is rotationally disordered and has been modeled as occupying two sites related by a 13 (1) degree rotation about the C8--C9 bond.

1-(4-iodobenzoyl)-5-methoxy-2-methyl-3-indoleacetic acid, an iodinated indomethacin analog.

The crystal structure of 1-(4-iodobenzoyl)-5-methoxy-2-methyl-3-indoleacetic acid, C19H16INO4, an analog of indomethacin, is reported. Bond distances and angles in the title compound closely resemble those reported for indomethacin and reflect the presence of steric strain at the site of the linkage between the 4-iodobenzoyl group and the indole moiety. The orientation of the 4-iodobenzoyl group with respect to the indole ring is not the same in the title compound as it is in indomethacin; the two structures are related by a rotation of 186 degrees about the C2--N1--C10--C11 torsion angle.

Probing the roles of active site residues in D-xylose isomerase.

The roles of active site residues His54, Phe94, Lys183, and His220 in the Streptomyces rubiginosus D-xylose isomerase were probed by site-directed mutagenesis. The kinetic properties and crystal structures of the mutant enzymes were characterized. The pH dependence of diethylpyrocarbonate modification of His54 suggests that His54 does not catalyze ring-opening as a general acid. His54 appears to be involved in anomeric selection and stabilization of the acyclic transition state by hydrogen bonding. Phe94 stabilizes the acyclic-extended transition state directly by hydrophobic interactions and/or indirectly by interactions with Trp137 and Phe26. Lys183 and His220 mutants have little or no activity and the structures of these mutants with D-xylose reveal cyclic alpha-D-xylopyranose. Lys183 functions structurally by maintaining the position of Pro187 and Glu186 and catalytically by interacting with acyclic-extended sugars. His220 provides structure for the M2-metal binding site with properties which are necessary for extension and isomerization of the substrate. A second M2 metal binding site (M2') is observed at a relatively lower occupancy when substrate is added consistent with the hypothesis that the metal moves as the hydride is shifted on the extended substrate.

Bay- and fjord-region distortions in dibenz[a,j]anthracene and tetrabenzo[de,hi,mn,qr]naphthacene.

The crystal structure of 7,14-dimethyldibenz[a,j]anthracene (DMDBA) has been determined, and the crystal structure of tetrabenzo[de,hi,mn,qr]naphthacene (TBNC) has been redetermined at higher precision than previously reported. These molecules are polycyclic aromatic hydrocarbons (PAHs) that have, respectively, two hindered bay regions and two fjord regions; the former PAH is a known carcinogen. The extensive out-of-plane bending as a result of steric overcrowding in the bay and fjord regions in these PAHs is shown by these studies. For DMDBA, the angle between the 14-methyl group and the outer rings is 32.6 degrees. For TBNC, the angle between the outer rings of the molecule is 31.9 degrees. These structures are compared with those of related structures of 7,12-dimethylbenz[a]anthracene and dibenzo[g,p]chrysene. It appears that steric overcrowding in such PAHs can cause distortions of up to 33 degrees C. Such steric overcrowding will affect the conformations of bay- and fjord-region diolepoxides, which are the presumed activated metabolites in the carcinogenic process.