Glycosynthase-based synthesis of xylo-oligosaccharides using an engineered retaining xylanase from Cellulomonas fimi

Glycosynthases are synthetic enzymes derived from retaining glycosidases in which the catalytic nucleophile has been replaced. The mutation allows irreversible glycosylation of sugar acceptors using glycosyl fluoride donors to afford oligosaccharides without any enzymatic hydrolysis. Glycosynthase technology has proven fruitful for the facile synthesis of useful oligosaccharides, therefore the expansion of the glycosynthase repertoire is of the utmost importance. Herein, we describe for the first time a glycosynthase, derived from a retaining xylanase, that synthesizes a range of xylo-oligosaccharides. The catalytic domain of the retaining endo-1,4-beta-xylanase from Cellulomonas fimi (CFXcd) was successfully converted to the corresponding glycosynthase by mutation of the catalytic nucleophile to a glycine residue. The mutant enzyme (CFXcd-E235G) was found to catalyze the transfer of a xylobiosyl moiety from alpha-xylobiosyl fluoride to either p-nitrophenyl beta-xylobioside or benzylthio beta-xylobioside to afford oligosaccharides ranging in length from tetra- to dodecasaccharides. These products were purified by high performance liquid chromatography in greater than 60% combined yield. 1H and 13C NMR spectroscopic analyses of the isolated p-nitrophenyl xylotetraoside and p-nitrophenyl xylohexaoside revealed that CFXcd-E235G catalyzes both the regio- and stereo-selective synthesis of xylo-oligosaccharides containing, exclusively, beta-(1 --> 4) linkages.     

Hydrogen bonding in the mechanism of GDP-mannose mannosyl hydrolase

GDP-mannose mannosyl hydrolase (GDPMH) from E. coli catalyzes the hydrolysis of GDP-α-d-sugars to GDP and β-d-sugars by nucleophilic substitution with inversion at the anomeric C1 of the sugar, with general base catalysis by His-124. The 1.3 Å X-ray structure of the GDPMH-Mg²⁺-GDP complex was used to model the complete substrate, GDP-mannose into the active site. The substrate is linked to the enzyme by 12 hydrogen bonds, as well as by the essential Mg²⁺. In addition, His-124 was found to participate in a hydrogen bonded triad: His-124-NδHTyr-127-OHPro-120(CO). The contributions of these hydrogen bonds to substrate binding and to catalysis were investigated by site-directed mutagenesis. The hydrogen bonded triad detected in the X-ray structure was found to contribute little to catalysis since the Y127F mutation of the central residue shows only 2-fold decreases in both k_cat and K_m. The GDP leaving group is activated by the essential Mg²⁺ which contributes at least 10⁵-fold to k_cat, and by nine hydrogen bonds, including those from Tyr-103, Arg-37, Arg-52, and Arg-65 (via an intervening water), each of which contribute factors to k_cat ranging from 24- to 309-fold. Both Arg-37 and Tyr-103 bind the β-phosphate of the leaving GDP and are only 5.0 Å apart. Accordingly, the R37Q/Y103F double mutant shows partially additive effects of the two single mutants on k_cat, indicating cooperativity of Arg-37 and Tyr-103 in promoting catalysis. The extensive activation of the GDP leaving group suggests a mechanism with dissociative character with a cationic oxocarbenium-like transition state and a half-chair conformation of the sugar ring, as found with glycosidase enzymes. Accordingly, Asp-22 which contributes 10^2.1- to 10^2.6-fold to k_cat, is positioned to both stabilize a developing cationic center at C1 and to accept a hydrogen bond from the C2–OH of the mannosyl group, and His-88, which contributes 10^2.3-fold to k_cat, is positioned to accept a hydrogen bond from the C3–OH of the mannose facilitating its distortion to a half-chair conformation. Also, the fluorinated substrate GDP-2-fluoro-α-d-mannose, for which the oxocarbenium ion-like transition state centered at C1 would be destabilized by electron withdrawal, shows a 16-fold lower k_cat and a 2.5-fold greater K_m than does GDP-α-d-mannose. The product of the contributions to catalysis of Arg-37 and Tyr-103 (taking their cooperativity into account), Arg-52, Arg-65, Mg²⁺, Asp-22, His-124, and His-88 is ≥10¹⁹, which exceeds the 10¹²-fold rate acceleration produced by GDPMH by a factor ≥10⁷. Hence, additional pairs or groups of catalytic residues must act cooperatively to promote catalysis.     

Structure, crystal chemistry and thermal evolution of the δ-Bi2O3-related phase Bi9ReO17

The thermal evolution and structural properties of fluorite-related δ-Bi₂O₃-type Bi₉ReO₁₇ were studied with variable temperature neutron powder diffraction, synchrotron X-ray powder diffraction and electron diffraction. The thermodynamically stable room-temperature crystal structure is monoclinic P2₁/c, a=9.89917(5), b=19.70356(10), c=11.61597(6) Å, β=125.302(2)° (R_p=3.51%, wR_p=3.60%) and features clusters of ReO₄ tetrahedra embedded in a distorted Bi-O fluorite-like network. This phase is stable up to 725 °C whereupon it transforms to a disordered δ-Bi₂O₃-like phase, which was modeled with δ-Bi₂O₃ in cubic Fm3¯m with a=5.7809(1) Å (R_p=2.49%, wR_p=2.44%) at 750 °C. Quenching from above 725 °C leads to a different phase, the structure of which has not been solved but appears on the basis of spectroscopic evidence to contain both ReO₄ tetrahedra and ReO₆ octahedra.     

Synthesis and structural studies of lanthanide substituted bismuth-titanium pyrochlores

The identity of the pyrochlore phase seen during the synthesis of ferroelectric Bi_4−xLn_xTi₃O₁₂ Aurivillius oxides is shown to be Bi_2/3Ln_4/3Ti₂O₇. This pyrochlore is only stable for Ln³⁺=Sm³⁺ or smaller. For larger lanthanides the layered Aurivillius oxide is favoured. The presence of six-fold disorder, associated with the Bi 6s² lone pair electrons, is believed to stabilise the unexpected stoichiometry of this oxide. Precise structures, obtained by Rietveld refinement from synchrotron X-ray diffraction data, of three examples Ln³⁺=Eu, Ho and Yb are presented.     

Design, construction, and validation of a 1-mm triple-resonance high-temperature-superconducting probe for NMR

We report a 600-MHz 1-mm triple-resonance high-temperature-superconducting (HTS) probe for nuclear magnetic resonance spectroscopy. The probe has a real sample volume of about 7.5 microl, an active volume of 6.3 microl, and appears to have the highest mass sensitivity at any field strength. The probe is constructed with four sets of HTS coils that are tuned to 1H, 2H, 13C, and 15N, and there is a z-axis gradient. The coils are cooled with a conventional Bruker CryoPlatform to about 20 K, and the sample chamber can be regulated above or below room temperature over a moderate range using a Bruker variable temperature unit. The absolute S/N for 0.1% ethylbenzene is approximately 1/3 that of a conventional 5mm probe with just 1/70 of the sample volume. We demonstrate the utility of this probe for small molecules and proteins with 2D spectra of just 1.7 microg of ibuprofen and 400 microM 15N-labeled ubiquitin.     

Expansions for the multivariate normal

Mehler gave an expansion for the standard bivariate normal density. Kibble extended it to a multivariate normal density whose covariance is a correlation matrix. We give extensions of these expansions for general covariances.     

Nature's many mechanisms for the degradation of oligosaccharides

Recent work on the mechanistic elucidation of the polysaccharide lyases, the [small alpha]-1,4-glucan lyases, and the Family 4 glycosidases have demonstrated that nature has evolved to use elimination steps for the degradation of oligosaccharides. The polysaccharide lyases (E.C. 4.2.2.-) have been shown to cleave uronic acid-containing polysaccharides via a stepwise E1cB mechanism. The mechanism of the alpha-1,4-glucan lyases (E.C. 4.2.2.13) is similar to the Family 31 glycosidases, forming a covalent glycosyl-enzyme intermediate, which is subsequently cleaved by an E1-like E2 mechanism. Meanwhile, the Family 4 glycosidases (E.C. 3.2.1.6) are suggested to undergo an oxidation-elimination-addition-reduction sequence. These three groups of enzymes are examples of stark contrast to the vast number of well-characterized glycosidases (E.C. 3.2.1.-), which utilize either the direct or double displacement mechanisms as proposed by Koshland over 50 years ago.     

Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: tyrosine is the catalytic nucleophile

Modified sialic acid substrates have been used to label Trypanosoma cruzi trans-sialidase, demonstrating that the enzyme catalyses the transfer of sialic acid through a covalent glycosyl-enzyme intermediate, a mechanism common to most retaining glycosidases. Peptic digestion of labeled protein, followed by LC-MS/MS analysis of the digest, identified Tyr342 as the catalytic nucleophile. This is the first such example of a retaining glycosidase utilizing an aryl glycoside intermediate. It is suggested that this alternative choice of nucleophile is a consequence of the chemical nature of sialic acid. A Tyr/Glu couple is invoked to relay charge from a remote glutamic acid, thereby avoiding electrostatic repulsion with the sialic acid carboxylate group.     

A xylobiose-derived isofagomine lactam glycosidase inhibitor binds as its amide tautomer

The atomic-resolution structure of a xylobiose-derived isofagomine lactam in complex with the xylanase Xyn10A from Streptomyces lividans reveals that the lactam is bound to the enzyme as the amide tautomer, with "reversed" protonation-states for nucleophile and acid-base.