Metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae. Participation and properties of sucrose-6-phosphate hydrolase and phospho-alpha-glucosidase.

Klebsiella pneumoniae is presently unique among bacterial species in its ability to metabolize not only sucrose but also its five linkage-isomeric alpha-d-glucosyl-d-fructoses: trehalulose, turanose, maltulose, leucrose, and palatinose. Growth on the isomeric compounds induced a protein of molecular mass approximately 50 kDa that was not present in sucrose-grown cells and which we have identified as an NAD(+) and metal ion-dependent 6-phospho-alpha-glucosidase (AglB). The aglB gene has been cloned and sequenced, and AglB (M(r) = 49,256) has been purified from a high expression system using the chromogenic p-nitrophenyl alpha-glucopyranoside 6-phosphate as substrate. Phospho-alpha-glucosidase catalyzed the hydrolysis of a wide variety of 6-phospho-alpha-glucosides including maltose-6'-phosphate, maltitol-6-phosphate, isomaltose-6'-phosphate, and all five 6'-phosphorylated isomers of sucrose (K(m) approximately 1-5 mm) yet did not hydrolyze sucrose-6-phosphate. By contrast, purified sucrose-6-phosphate hydrolase (M(r) approximately 53,000) hydrolyzed only sucrose-6-phosphate (K(m) approximately 80 microm). Differences in molecular shape and lipophilicity potential between sucrose and its isomers may be important determinants for substrate discrimination by the two phosphoglucosyl hydrolases. Phospho-alpha-glucosidase and sucrose-6-phosphate hydrolase exhibit no significant homology, and by sequence-based alignment, the two enzymes are assigned to Families 4 and 32, respectively, of the glycosyl hydrolase superfamily. The phospho-alpha-glucosidase gene (aglB) lies adjacent to a second gene (aglA), which encodes an EII(CB) component of the phosphoenolpyruvate-dependent sugar:phosphotransferase system. We suggest that the products of the two genes facilitate the phosphorylative translocation and subsequent hydrolysis of the five alpha-d-glucosyl-d-fructoses by K. pneumoniae.

Phosphorylation and metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae.

Not only sucrose but the five isomeric alpha-D-glucosyl-D-fructoses trehalulose, turanose, maltulose, leucrose, and palatinose are utilized by Klebsiella pneumoniae as energy sources for growth, thereby undergoing phosphorylation by a phosphoenolpyruvate-dependent phosphotransferase system uniformly at 0-6 of the glucosyl moiety. Similarly, maltose, isomaltose, and maltitol, when exposed to these conditions, are phosphorylated regiospecifically at O-6 of their non-reducing glucose portion. The structures of these novel compounds have been established unequivocally by enzymatic analysis, acid hydrolysis, FAB negative-ion spectrometry, and 1H and 13C NMR spectroscopy. In cells of K. pneumoniae, hydrolysis of sucrose 6-phosphate is catalyzed by sucrose 6-phosphate hydrolase from Family 32 of the glycosylhydrolase superfamily. The five 6'-O-phosphorylated alpha-D-glucosyl-fructoses are hydrolyzed by an inducible (approximately 49-50 Kda) phospho-alpha-glucosidase from Family 4 of the glycosylhydrolase superfamily.

A history of research on yeasts 3: Emil Fischer, Eduard Buchner and their contemporaries, 1880-1900.

Through the discovery of Buchner, Biology was relieved of another fragment of mysticism. The splitting up of sugar into CO2 and alcohol is no more the effect of a "vital principle" than the splitting up of cane sugar by invertase. (Jacques Loeb 1906 [138] p.22.)

Structure and lipophilicity profile of 2,3-anhydro-alpha-cyclomannin and its ethanol inclusion complex.

Readily available from alpha-cyclodextrin in three steps, 2,3-anhydro-alpha-cyclomannin composed of six alpha-(1-->4)-linked 2,3-anhydro-D-mannopyranose residues, crystallizes well when precipitated from aqueous ethanol. An X-ray structure reveals the macrocycle to contain ethanol in its cavity, thus representing the first inclusion complex of a non-glucose cyclooligosaccharide. The wider rim of the torus-shaped macrocycle holds the six epoxide rings whose oxygens point away from the cavity, thereby sculpturing the unique over-all shape of a six-pointed star.

Topography of the 1:1 alpha-cyclodextrin-nitromethane inclusion complex.

Dissolution of alpha-cyclodextrin (alpha-CD) in 9:1 water-nitromethane smoothly generates the title compound, which crystallizes as the pentahydrate in the orthorhombic space group P2(1)2(1)2(1) with a = 9.452(4), b = 14.299(3), c = 37.380(10) A, and Z = 4. Its crystal structure analysis revealed the alpha-CD macrocycle in an unstrained conformation stabilized through a ring of O-2...O-3' hydrogen bonds between five of the six adjacent glucose residues. The nitromethane is located in the alpha-CD cavity in an orientation parallel to the plane of the macrocycle, and assumes two sites of equal population with the nitro group in excessive thermal motion; the guest is held by van der Waals contacts and C-H...O-type hydrogen bonds to the pyranose H-3 and H-5 protons. The packing of the macrocycles in the crystal lattice is of cage herringbone-type with an extensive intra- and intermolecular hydrogen bonding network. The ready formation of a nitromethane inclusion complex in aqueous nitromethane, and the subtleties of its molecular structure amply demonstrate the ease with which water is expelled from the alpha-CD cavity by a more hydrophobic co-solvent.

Cyclofructins with six to ten beta-(1-->2)-linked fructofuranose units: geometries, electrostatic profiles, lipophilicity patterns, and potential for inclusion complexation.

Cyclofructins composed of six (1, "CF6") to ten (5, "CF10") beta-(1-->2)-linked fructofuranose units were subjected to conformational analysis using Monte Carlo simulations based on the PIMM91 force-field. Breaking the molecular symmetry partially by alternating inclination of the spiro-type anellated fructofuranoses relative to the crown ether ring core, i.e. the 3-OH groups pointing either towards or away from the molecular center, substantially lowers the strain energy of the cyclofructins. The global energy-minimum geometries of CF6, CF8, and CF10 exhibit Cn/2 rotational symmetry, whilst the odd-membered macrocycles in CF7 and CF9 adopt C1 symmetry. Identical conformations of the solid-state geometry of CF6 (1) and its computer-generated form manifest the reliability of the computational analysis. The molecular surfaces calculated for the energy-minimum structures establish a disk-type shape for CF6 (1), CF7 (2), and CF8 (3), whereas further ring enlargement to CF9 (4) and CF10 (5) leads to torus-shaped molecules with through-going cavities. Color-coded projection of the molecular lipophilicity patterns (MLPs) and the electrostatic potential profiles (MEPs) onto these surfaces cogently displays the crown ether-like properties, favoring the complexation of metal cations via strong electrostatic interactions through the 3-OH groups located on the hydrophilic molecular side. The central cavities of CF9 and CF10 are characterized not only by significantly enhanced hydrophobicity, but also by highly negative electrostatic potentials around the narrow aperture of the tori made up by the 3-OH/4-OH groups, and positive potentials on the opposite rim. Accordingly, CF9 and CF10 are capable to form inclusion complexes, the cavity of the latter being approximately as large as the one of alpha-cyclodextrin. Calculation of the inclusion complex geometries of CF9 with beta-alanine and of CF10 with p-aminobenzoic acid revealed the guest to be deeply incorporated into the respective cavities, masking the guest's hydrophobic parts. Analysis of the electrostatic interactions at the interface of the zwitter-ionic guests with the oppositely polarized hosts predicts a high degree of regiospecificity for complex formation.

Incorporation of 1-deoxy-D-xylulose into isoprene and phytol by higher plants and algae.

In further substantiating the novel mevalonate-independent pathway for isoprenoid biosynthesis, which generates isopentenyl diphosphate (IPP) via 1-deoxy-D-xylulose-5-phosphate, labeling experiments with 1-[2H(1)]deoxy-D-xylulose were performed with various higher plants and algae: efficient incorporation was observed into isoprene emitted by Populus, Chelidonium, and Salix, into the phytol moiety of chlorophylls in a red alga (Cyanidium), in two green algae (Scenedesmus, Chlamydomonas), and a higher plant (Lemna). By contrast, 13C-mevalonate applied was incorporated into isoprene and phytol to a much lower extent or not at all. This demonstrates that this '1-deoxy-D-xylulose-5-phosphate pathway' for biosynthesis of plastidic isoprenoids is widely distributed in photosynthetic organisms.

4,6-di-O-benzoyl-3-O-benzyl-alpha-D-arabino-hexo-pyranos-2-ulosyl bromide: a conveniently accessible glycosyl donor for the expedient construction of diantennary beta-D-mannosides branched at O-3 and O-6.

A concise practical, large scale-adaptable six-step sequence has been developed for the transformation of diacetone-glucose into 4,6-di-O-benzoyl-3-O-benzyl-alpha-D-arabino-hexopyranos-2-ulosy l bromide (7), a most useful indirect beta-D-mannosyl donor as its blocking group pattern allows the construction of biologically relevant beta-D-mannosides branched at O-3 and O-6. The broad utility of this new ulosyl bromide 7 resides in its high anomeric reactivity, and in the ease and uniformity with which beta-stereocontrol can be achieved over both, glycosidations and carbonyl reduction of the beta-ulosides formed: Koenigs-Knorr conditions exclusively provide beta-glycosiduloses, hydride reduction of their carbonyl functions proceeds with high stereoselectivities (> 20:1) in favor of the beta-D-mannosides. These preparatively auspicious properties are materialized in an efficient, straightforward synthesis of alpha-D-Manp-(1-->6)-[alpha-D-Manp-(1-->3)]-beta-D-Manp++ +-(1-->O)-Octyl, the 3,6-O-branched core-mannotrioside carrying an octyl spacer instead of the chitobiosyl unit.

Solid-state conformations of 2,6-cis- and 2,6-trans-substituted dihydropyran-3-ones.

2,6-Dihydropyran-3-ones carrying substituents at C-2 and C-6 in cis-arrangement invariably adopt half-chair conformations in which the ring oxygen and the carbon atom next to the carbonyl group are above and below, respectively, the plane formed by the other four carbon atoms, i.e., the 2Ho or oH2 conformation. In the case of a trans-arrangement of 2,6-substituents, the geometry of the pyranoid ring falls into the Bo,6<-->Eo<-->2Ho or thet inverse o,6B<-->oE<-->oH2 section of the conformational cycle, depending on the absolute configuration of the compound; for two of the dihydropyranones, 4B and 6, a unique skew-boat (SBo,6) conformation, fixed between the Bo,6 and Eo geometries, was ascertained, which previously has only been observed for pyranoid enelactones.