Skew-boat Conformation Research Articles

Structure of the Humicola insolens cellobiohydrolase Cel6A D416A mutant in complex with a non-hydrolysable substrate analogue, methyl cellobiosyl-4-thio-beta-cellobioside, at 1.9 A.

The enzymatic degradation of cellulose continues to be one of the most important enzyme-catalysed reactions. Glycoside hydrolases from family GH-6 hydrolyse cellulose with inversion of the configuration of the anomeric carbon. Whilst the catalytic proton donor has been clearly identified (Asp226 in Humicola insolens Cel6A), the identification and even the existence of a potential Brønsted base remains unclear. Equally controversial is the role of surface-loop flexibility. Here, the structure of the D416A mutant of the H. insolens cellobiohydrolase Cel6A in complex with a non-hydrolysable thiooligosaccharide methyl cellobiosyl-4-thio-beta-cellobioside at 1.9 A resolution is presented. Substrate distortion in the -1 subsite, to a (2)S(0) skew-boat conformation, is observed, similar to that seen in the analogous Trichoderma reesei Cel6A structure [Zou et al. (1999), Structure, 7, 1035-1045], but the active-centre N-terminal loop of the H. insolens enzyme is found in a more open conformation than described for previous structures.

A Conformational Study on the Algal Polysaccharide Ulvan

The conformational behavior of the algal polysaccharide ulvan in aqueous solution has been studied. This biopolymer, containing limited regular regions of aldobiuronic sequences made of β-d-glcpA(1→4)-α-l-rhap 3-sulfate and of α-l-idopA(1→4)-α-l-rhap 3-sulfate, can be considered as a low-cost source of rare sugars with a potential impact on several industrial applications, from pharmaceutical to fine chemistry industry. The relationship of structure with the chemical composition of the saccharidic residues in the biopolymer is evidenced with respect to the ability of iduronic acid moiety to assume two main ring conformations, namely the 1C4 chair and the 2S0 skew boat conformations. Experimental results are interpreted in the light of models obtained from conformational analysis calculations.

Effect of peracylation of beta-cyclodextrin on the molecular structure and on the formation of inclusion complexes: an X-ray study.

The molecular structures of peracylated beta-cyclodextrins (CDs)--heptakis(2,3,6-tri-O-acetyl)-beta-CD (TA), heptakis(2,3,6-tri-O-propanoyl)-beta-CD (TP), and heptakis(2,3,6-tri-O-butanoyl)-beta-CD (TB)--have been determined by single crystal X-ray structure analysis. Due to the lack of O2...O3' hydrogen bonds between adjacent glucose units of the peracylated CDs, the macrocycles are elliptically distorted into nonplanar boat-shaped structures. The glucose units are tilted with respect to the O4 plane to relieve steric hindrance between adjacent acyl chains. In TB, all glucose units adopt the common (4)C(1)-chair conformation and one butanoyl chain intramolecularly penetrates the cavity, whereas, in TA and TP, one glucose unit each occurs in (O)S(2)-skew-boat conformation and one acyl chain closes the O6 side like a lid. In each of the three homologous molecules the intramolecular self-inclusion and lidlike orientation of acyl chains forces the associated O5-C5-C6-O6 torsion angle into a trans-conformation never observed before for unsubstituted CD; the inclusion behavior of TA, TP, and TB in solution has been studied by circular dichroism spectroscopy with the drug molsidomine and several organic compounds. No inclusion complexes are formed, which is attributed to the intramolecular closure of the molecular cavity by one of the acyl chains.

Atomic resolution structure of endoglucanase Cel5A in complex with methyl 4,4II,4III,4IV-tetrathio-alpha-cellopentoside highlights the alternative binding modes targeted by substrate mimics.

Many three-dimensional structures of retaining beta-D-glycoside hydrolases have been determined, yet oligosaccharide complexes in which the ligand spans the catalytic centre are rare. Those that have been reported so far have revealed two modes of binding: those in which the substrate adopts a distorted skew-boat or envelope conformation in the -1 subsite, reflecting the distortion observed during the catalytic cycle, and those which bypass the true catalytic centre and thus lie in a non-productive manner across the -1 subsite. The three-dimensional structure of a retaining endocellulase, Bacillus agaradhaerens Cel5A, in complex with methyl 4,4(II),4(III),4(IV)-tetrathio-alpha-cellopentoside falls into this latter category. The 1.1 A structure reveals the binding of five pyranosides, all in the (4)C(1) chair conformation, occupying the -3, -2, +1 and +2 subsites whilst evading the catalytic machinery located in the true -1 subsite. Such binding is in marked contrast to the structure of another retaining endocellulase, the Fusarium oxysporum Cel7B, the identical ligand in which displayed a distorted skew-boat conformation at the active centre. These two binding modes may reflect different steps in the binding and catalytic process.

Synthesis of Conformationally Locked Carbohydrates: A Skew-Boat Conformation of L-Iduronic Acid Governs the Antithrombotic Activity of Heparin

Synthesis of Conformationally Locked Carbohydrates: A Skew-Boat Conformation of L-Iduronic Acid Governs the Antithrombotic Activity of Heparin

Mechanisms of glycosyl transferases and hydrolases

Glycosidases and glycosyl transferases fall into two major mechanistic classes; those that hydrolyse the glycosidic bond with retention of anomeric configuration and those that do so with inversion. There are, however, two classes of transferases: those that use nucleotide phosphosugars (NP-sugar-dependent) and those that simply transglycosylate between oligosaccharides or polysaccharides (transglycosylases). The latter are mechanistically similar to retaining glycosidases while the mechanisms of NP-sugar-dependent transferases are far from clear. Retaining glycosidases and the transglycosylases employ a mechanism involving a covalent glycosyl–enzyme intermediate formed and hydrolysed with acid/base catalytic assistance via oxocarbenium ion-like transition states. This intermediate has been trapped on glycosidases in two distinct ways, either by modification of the substrate through fluorination, or of the enzyme through mutation of key residues. A third method has been developed for trapping the intermediate on transglycosylases involving the use of incompetent substrates that allow formation of the intermediate, but prohibit its transfer as a consequence of their acceptor hydroxyl group being removed. Three-dimensional structures of several of these glycosyl–enzyme complexes, along with those of Michaelis complexes, have been determined through X-ray crystallographic analysis, revealing the identities of important amino acid residues involved in catalysis. In particular they reveal the involvement of the carbonyl oxygen of the catalytic nucleophile in strong hydrogen bonding to the sugar 2-hydroxyl for the β-retainers or in interactions with the ring oxygen for α-retainers. The glucose ring in the −1 (cleavage) site in the intermediates formed on several cellulases and a β-glucosidase adopts a normal 4C 1 chair conformation. By contrast the xylose ring at this site in a xylanase is substantially distorted into a 2,5B boat conformation, an observation that bears significant stereoelectronic implications. Substantial distortion is also observed in the substrate upon binding to several β-glycosidases, this time to a 1S 3 skew boat conformation. Much less distortion is seen in the substrate bound on an α-transglycosylase. Finally an efficient catalyst for synthesis, but not hydrolysis, of glycosidic bonds has been generated by mutation of the glutamic acid catalytic nucleophile of a β-glucosidase to an alanine. When used with α-glucosyl fluoride as a glycosyl donor, along with a suitable acceptor, oligosaccharides up to five sugars in length have been made with yields of up to 90% on individual steps. These new enzymes have been named Glycosynthases.

3-(2-Furyl)-6-(4-methylphenyl)-7H-1,2,4-triazolo[3,4-b][1,3,4]thiadiazine and its 6-phenyl analogue

In the title compounds, C15H12N4OS, (I), and C14H10N4OS, (II), the thia­diazine ring adopts a skew-boat conformation, while the triazole and furyl rings are essentially planar. The phenyl group is twisted by 33.5 (2) and 47.9 (1)° out of the triazole-ring plane in (I) and (II), respectively.

Organoindium Thiolate Oligomers: An X-ray Structural Study

The reaction of R3In (R = methyl, tert-butyl, xylyl) with bis(tricyclohexyltin) sulfide, 2-methylpropane-2-thiol, 4,6-dimethyl-2-mercaptopyrimidine, cyclopentanethiol, cyclohexanethiol, 2,5-dimethylbenzenethiol, or 2,6-methylbenzenethiol has yielded seven new indium−sulfur complexes: {m-Xyl2In[μ-SSn(C6H11)3]}2 (2), [t-Bu2In(μ-S-C5H9)]2 (3), [t-Bu2In(μ-S-C6H11)]2 (4), {t-Bu2In[μ-S(2,5-Me2)C6H3]}2 (5), [Me2In(μ-S-t-Bu)]3 (6), {t-Bu2In[μ-S(4,6-Me2)C4N2H]}3 (7), and {Me2In[μ-S(2,6-Me2)C6H3]}4 (8). These complexes have been studied by 1H and 13C NMR spectroscopy, and their structures have been determined by single-crystal X-ray diffraction techniques. 2−5 form dimeric species with planar (In−S)2 cores, 6 forms a trimer which adopts a skew-boat conformation, 7 forms a trimer with a planar six-membered (InS)3 ring with a secondary In−N interaction, giving rise to a pseudo-five-coordinate In atom, and 8 forms a tetramer in an extended chair conformation, the first known indium tetramer. Several of the factors of...

(E)-1,2-Bis(3,4-dihydro-1-methoxynaphthalen-2-yl)ethylene

In the structure of the title compound, C 24 H 24 O 2 , one of the two types of six-membered rings is planar and the other is in a skew-boat conformation. The ethylene C=C bond distance is 1.345 (3) A. The substituents at the ethylene are in an E configuration. The crystal structure consists of discrete molecules lying about inversion centres, held together by van der Waals contacts.

Methyl (5SR,6SR)-1,3-dichloro-8-diethylamino-5,6-dihydro-5-hydroxy-7-nitro-6-phenylisoquinoline-5-carboxylate

In the title compound, C 21 H 21 Cl 2 N 3 O 5 , the fused cyclohexadiene ring adopts a skew-boat conformation, with the methoxycarbonyl and phenyl groups attached in axial positions and the nitro and diethylamino groups attached equatorially. The mean plane through the cyclohexadiene ring forms dihedral angles of 23.30(7) and 87.72(8)° with the pyridine and phenyl rings, respectively. In the crystal, the inversion-related molecules exist as O-H… O hydrogen-bonded dimers.

Trimethyl (5SR,6SR)-1,3-dichloro-8-diethylamino-5,6-dihydro-5-hydroxyisoquinoline-5,6,7-tricarboxylate

The title compound, C 19 H 22 C 12 N 2 O 7 , has four independent molecules in the asymmetric unit with no pseudosymmetry. In each of the four molecules the fused cyclohexadiene ring adopts a skew-boat conformation, with two of the three methoxycarbonyl groups in the axial orientation and the third methoxycarbonyl and the diethylamino groups attached equatorially. The hydroxyl groups are involved in O-H…O intermolecular hydrogen bonds with carbonyl-O atoms to form infinite chains in the [210] direction.

Solid-State Modeling. VI. 2,3-Diketopiperazine: the Integration of Crystallographic and Spectroscopic Evidence

2,3-Diketopiperazine (2,3-piperazinedione) crystallizes in the monoclinic space group P21/c with a = 5.941 (3), b = 10.080 (3), c = 8.282 (2) Å and β = 95.87 (3)°. The six-membered ring adopts a skew-boat conformation with Q = 0.467 (3) Å, θ = 64.6 (3)° and φ = 269.8 (4)°. Ab initio calculations show that the perfect skew-boat with its C 2 symmetry is broken by the formation of two intermolecular N—H...O bonds, involving only one of the C=O groups of the 2,3-diketopiperazine molecule. Vibrational spectra were recorded in solution and in the solid state. The assignment of the normal vibrations is proposed based on comparison with spectra of similar molecules and spectral changes due to deuteration. Ab initio calculations for the isolated molecule and the solid-state structure were used to calculate differences in the molecular geometry in the gas phase and crystalline state. Using these reference structures we calculated the stretching frequencies for the C=O groups. We predict an IR shift for C=O of 130 cm−1, when the molecule goes from the gas phase to the solid state. The observed shift is 110 cm−1. The differences between the C=O moieties in the solid state produce a calculated Δν of 55 cm−1, which matches satisfactorily the observed value of 49 cm−1.

Net Intensities: Accuracy Improvement Through a Bayesian Perspective on the Measuring Strategy and Their Persistent Lack of Precision. An Illustration

2,3-Diketopiperazine (2,3-piperazinedione), C4H6N2O2, crystallizes in the monoclinic space group P21/c with a = 5.941 (3), b = 10.080 (3), c = 8.282 (2) Å, β = 95.87 (3)°, V = 493.4 (5) Å3, Z = 4, Dx = 1.536 Mg m−3 and Mr = 114.1. The six-membered ring adopts a skew-boat conformation with Q = 0.467 (3) Å, θ = 64.6 (3)° and φ = 269.8 (4)°. The C 2 symmetry, which typifies the free molecule, is broken by the formation of two intermolecular N—H...O bonds involving only one of the C=O groups of the 2,3-diketopiperazine molecule. The intensity data typical for a time series are summarized in traditional statistics by B = σ2(B) = ΣBi and R = σ2(R) = ΣRi , where B is the background and R the raw intensity. Exploitation of the same data using Bayesian methodology leads to similar values for B and R, but the variances for these signals are significantly smaller. This reduction in variance is dictated by the length N of the time series. With 25 observations in each hkl time series we arrive at variances that are 25% of their classical values. So, a measuring strategy in which a single observation with a slow scan at speed 1/N is preferred above a series of N observations at speed 1 produces the worst possible benefit within a fixed time frame. In complete contrast to our expectation, structure refinements reveal that that the standard deviation of an observation of unit weight, S, will converge towards its ideal unit value only when we decrease the accuracy of the data set. This unpleasant behaviour points to a serious discrepancy between accuracy and precision. Our reflection intensities are systematically wrong, because we underestimate the wavelength dispersion. On our CAD-4 equipment a sealed Mo tube and the standard graphite monochromator produce an incident beam with Δλ/λ = 14%. As a consequence, the observed background intensities are not representative of the real background. The size of the background error depends on the nearby Bragg intensity. The magnitude of the signal pollution contaminating the local background is ∼2% of the maximum Bragg intensity found at λ = 0.71 Å.

Mechanism of heparin activation of antithrombin: evidence for an induced-fit model of allosteric activation involving two interaction subsites.

The anticoagulant activation of the serpin antithrombin by heparin pentasaccharide DEFGH was previously shown to involve trisaccharide DEF first binding and inducing activation of the serpin, followed by disaccharide GH binding and stabilizing the activated state [Petitou et al. (1997) Glycobiology 7, 323-327; Desai et al. (1998) J. Biol. Chem. 273, 7478-7487]. In the present study, the role of conformational changes and charged residues of the GH disaccharide in the allosteric activation mechanism was investigated with variant pentasaccharides modified in the GH disaccharide. Perturbation of the conformational equilibrium of iduronate residue G through replacement of the nonessential 3-OH of this residue with -H resulted in parallel decreases in the fraction of residue G in the skew boat conformer (from 64 to 24%) and in the association constant for pentasaccharide binding to antithrombin [(2.6 +/- 0.3)-fold], consistent with selective binding of the skew boat conformer to the serpin. Introduction of an additional sulfate group to the 3-OH of residue H flanking a putative charge cluster in the GH disaccharide greatly enhanced the affinity for the serpin by approximately 35-fold with only a small increase in the fraction of residue G in the skew boat conformation (from 64 to 85%). The salt dependence of binding, together with a recent X-ray structure of the antithrombin-pentasaccharide complex, suggested that the majority of the enhanced affinity of the latter pentasaccharide was due to direct electrostatic and hydrogen-bonding interactions of the H residue 3-O-sulfate with antithrombin. All variant pentasaccharides produced a normal enhancement of antithrombin fluoresence and normal acceleration of factor Xa inhibition by the serpin at saturating levels, indicating that conformational activation of antithrombin was not affected by the pentasaccharide modifications. Rapid kinetic studies were consistent with the altered affinities of the variant pentasaccharides resulting mostly from perturbed interactions of the reducing-end GH disaccharide with the activated antithrombin conformation and minimally to an altered binding of the nonreducing-end DEF trisaccharide to the native serpin conformation. Together, these results support a model in which the conformational flexibility of the G residue facilitates conversion to the skew boat conformer and thereby allows charged groups of the GH disaccharide to bind and stabilize the activated antithrombin conformation that is induced by the DEF trisaccharide.

Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase.

The enzymatic hydrolysis of O-glycosidic linkages is one of the most diverse and widespread reactions in nature and involves a classic "textbook" enzyme mechanism. A multidisciplinary analysis of a beta-glycoside hydrolase, the Cel5A from Bacillus agaradhaerens, is presented in which the structures of each of the native, substrate, covalent-intermediate, and product complexes have been determined and their interconversions analyzed kinetically, providing unprecedented insights into the mechanism of this enzyme class. Substrate is bound in a distorted 1S3 skew-boat conformation, thereby presenting the anomeric carbon appropriately for nucleophilic attack as well as satisfying the stereoelectronic requirements for an incipient oxocarbenium ion. Leaving group departure results in the trapping of a covalent alpha-glycosyl-enzyme intermediate in which the sugar adopts an undistorted 4C1 conformation. Finally, hydrolysis of this intermediate yields a product complex in which the sugar is bound in a partially disordered mode, consistent with unfavorable interactions and low product affinity.

Structure of the endoglucanase I from Fusarium oxysporum: native, cellobiose, and 3,4-epoxybutyl beta-D-cellobioside-inhibited forms, at 2.3 A resolution.

The mechanisms involved in the enzymatic degradation of cellulose are of great ecological and commercial importance. The breakdown of cellulose by fungal species is performed by a consortium of free enzymes, known as cellobiohydrolases and endoglucanases, which are found in many of the 57 glycosyl hydrolase families. The structure of the endoglucanase I (EG I), found in glycosyl hydrolase family 7, from the thermophilic fungus Fusarium oxysporum has been solved at 2.3 A resolution. In addition to the native enzyme, structures have also been determined with both the affinity label, 3,4-epoxybutyl beta-D-cellobioside, and the reaction product cellobiose. The affinity label is covalently bound, as expected, to the catalytic nucleophile, Glu197, with clear evidence for binding of both the R and S stereoisomers. Cellobiose is found bound to the -2 and -1 subsites of the enzyme. In marked contrast to the structure of EG I with a nonhydrolyzable thiosaccharide analog, which spanned the -2, -1, and +1 subsites and which had a skew-boat conformation for the -1 subsite sugar [Sulzenbacher, G., et al. (1996) Biochemistry 35, 15280-15287], the cellobiose complex shows no pyranoside ring distortion in the -1 subsite, implying that strain is induced primarily by the additional +1 subsite interactions and that the product is found, as expected, in its unstrained conformation.

Bis(ethylene) complexes of molybdenum- and tungsten-(0) containing the CH3C(CH2PMe2)3 ligand: synthesis, solution conformation and reactivity

The reaction of trans-[M(C2H4)2(PMe3)4] with the tripodal phosphine 1,1,1-tris[(dimethylphosphino)methyl]ethane (CP3) affords complexes trans-[M(C2H4)2(η2-CP3)(PMe3)2] (M = Mo 1a or W 1b). A detailed NMR study, including hetero- and homo-nuclear two-dimensional correlations, has been carried out for 1b which allows a complete assignment of signals and a structural spectroscopic determination. In solution, no ethylene rotation is observed at room temperature and the preferential conformer of the six-membered ring W[η2-CH3C(CH2PMe2)3] fragment is, on the basis of steric arguments, the skew-boat conformation. Interaction of compounds 1 with CO affords trans-[M(C2H4)2(η2-CP3)(CO)(PMe3)] (M = Mo 2a or W 2b), while reaction of 1a with CNBut furnishes the compound trans-[Mo(C2H4)2(η2-CP3)(CNBut)(PMe3)] 3. The bidentate co-ordination of CP3 induces chirality at the metal center and compounds 2 and 3 are obtained as a ca. 1∶1 mixture of diastereoisomers, identified by the presence of two different patterns of resonances in their 31P-{1H} NMR spectra at low temperature. At the fast regimen exchange, the isomers are interconverted through bis(ethylene) rotation. A variable-temperature 31P-{1H} NMR study carried out for 2a gives an approximate value of ΔG‡ for this process of 55 kJ mol–1 at 298 K.

Synthesis and reactivity of an unsymmetrically substituted 16-membered macrocyclic complex of nickel(II) and the crystal structure of its acyclic precursor, (3-acetyl-10-methyl-5,9-diazatrideca-3,8-diene-2, 12-dionato)nickel(II)

The synthesis of the new unsymmetrically substituted 16-membered macrocyclic complex, (3-acetyl-2,10,12-trimethyl-1, 5, 9, 13-tetraazacyclohexadeca-1, 3, 9, 11-tetraenato)-NiII, by template-controlled macrocyclization is reported. This complex has a single nucleophilic site available to react with electrophilic reagents. The crystal structure of the acyclic precursor, (3-acetyl-10-methyl-5,9-diazatrideca-3, 8-diene-2,12-dionato)NiII shows that in the solid state it exists as a pair of enantiomers, arising from the presence of two possible skew boat conformations adopted by the six-membered saturated metallochelate ring present in the molecule.

A Condensed Dialkoxide Complex of Copper(II), [(CH3)2NCH2CH2N(CH3)2]Cu[{OC(CF3)2}2O

The title complex, [1,1,1,5,5,5-hexa­fluoro-3-oxa-2,4-bis­(trifluoromethyl)-2,4-pentanediolato-O,O']- (N,N,N',N'-tetramethylethylenediamine-N,N')copper(II), [Cu(C6F12O3)(C6H16N2)], contains a condensed dialkoxide ligand forming a markedly non-planar six-membered chelate ring with a skew-boat conformation. There are two independent molecules in the unit cell. In molecule (1), the absolute conformations of the tetramethylethylenediamine and dialkoxide rings are both λ and in molecule 2, they are δ and λ, respectively. The mean O—Cu—O angle is 92.6 (3)°, with a mean Cu—O bond length of 1.893 (3) A.

The Dichloroplatinum(II) and Dichloropalladium(II) Complexes of 1,4-Diazacycloheptane: Preparative, Structural and Conformational Studies

The preparation and purification of [Pt( hpip )Cl2] and [Pd( hpip )Cl2] ( hpip = homopiperazine = 1,4-diazacycloheptane) are described. Crystal structures of [Pt( hpip )Cl2] and [Pd( hpip )Cl2] have been determined by X-ray diffraction methods and refined to R values of 0.023 (932 F) and 0.023 (948 F). The crystals of [Pt( hpip )Cl2] are orthorhombic, space group Pbcm , a 7.7019(8), b 9.8080(12), c 12.1944(14) Ǻ, and those of [Pd( hpip )Cl2] are monoclinic, space group P21/m, a 6.1001(9), b 11.527(2), c 6.458(I) Ǻ, β 106.30(2)°. The seven- membered rings of the ligands in both complexes adopt boat-like conformations in which the five- membered chelate ring has an eclipsed N-C-C-N group and the six- membered chelate ring adopts a chair conformation. Molecular mechanics methods were used to investigate whether this conformation was a crystallographic artefact but it was found to be real. An alternative conformation in which the six-membered chelate ring adopts a skew-boat conformation was also investigated. It was found to be less stable than the conformation observed in the crystal structures, but to a degree that depends on whether non-bonded interactions involving the metal atom were included or not.