A highly efficient deprotection of the 2,2,2-trichloroethyl group at the anomeric oxygen of carbohydrates

Commercially available zinc dust in the presence of ammonium chloride in acetonitrile at reflux removes the 2,2,2-trichloroethyl (TCE) group at anomeric centers with excellent yields (>95%) in short reaction times. This present method is easily implemented on substrates containing acyl and benzyl groups and large-scale reactions also proceed in high yield.     

Fucokinase, its anomeric specificity and mechanism of phosphate group transfer

Fucokinase phosphorylates L-fucose at the anomeric position and, as such, might use either the alpha or beta anomer as its substrate. Examination of the utilization of radiolabelled alpha and alpha,beta mixtures established beta-L-fucose as the required substrate. Phosphorylation at the anomeric center might involve either the loss or retention of the anomeric oxygen. The mechanism has been shown to involve anomeric oxygen retention through mass spectrometric analysis of the product phosphate derived from 18O-labelled L-fucose.     

Substrate-induced activation of maltose phosphorylase: interaction with the anomeric hydroxyl group of alpha-maltose and alpha-D-glucose controls the enzyme's glucosyltransferase activity

Maltose phosphorylase, long considered strictly specific for beta-D-glucopyranosyl phosphate (beta-D-glucose 1-P), was found to catalyze the reaction beta-D-glucosyl fluoride + alpha-D-glucose----alpha-maltose + HF, at a rapid rate, V = 11.2 +/- 1.2 mumol/(min.mg), and K = 13.1 +/- 4.4 mM with alpha-D-glucose saturating, at 0 degrees C. This reaction is analogous to the synthesis of maltose from beta-D-glucose 1-P + D-glucose (the reverse of maltose phosphorolysis). In acting upon beta-D-glucosyl fluoride, maltose phosphorylase was found to use alpha-D-glucose as a cosubstrate but not beta-D-glucose or other close analogs (e.g., alpha-D-glucosyl fluoride) lacking an axial 1-OH group. Similarly, the enzyme was shown to use alpha-maltose as a substrate but not beta-maltose or close analogs (e.g., alpha-maltosyl fluoride) lacking an axial 1-OH group. These results indicate that interaction of the axial 1-OH group of the disaccharide donor or sugar acceptor with a particular protein group near the reaction center is required for effective catalysis. This interaction appears to be the means that leads maltose phosphorylase to promote a narrowly defined set of glucosyl transfer reactions with little hydrolysis, in contrast to other glycosylases that catalyze both hydrolytic and nonhydrolytic reactions.     

On the role of the sterol hydroxyl group in membranes.

The adequacy of sterol derivatives containing a blocked 3-hydroxyl group for sustaining the growth of two sterol auxotrophs has been investigated. Mycoplasma capricolum, a cholesterol-requiring bacterium, grows nearly as well on media supplemented with cholesteryl methyl ether or cholesteryl acetate as on free cholesterol. The two derivatives are recovered unchanged from the bacterial cells. Similarly, cholesteryl methyl ether or ergosteryl methyl ether replace cholesterol or ergosterol as sterol sources for a yeast mutant, strain GL7, defective in 2,3-oxidosqualene-lanosterol cyclization. During aerobic or semianaerobic growth, yeast cells demethylate some of the cholesteryl methyl ether to free cholesterol. However, cells growing on cholesterol methyl ether under strict anaerobic conditions do not produce free sterol. The bearing of these results on the postulated requirement of a free sterol hydroxyl group for membrane function is discussed. Sterol esterification does not appear to be essential for the two microbial systems.     

MHC interaction and T cell recognition of carbohydrates and glycopeptides.

The T cell independence of complex polysaccharide Ag has suggested the possibility that carbohydrates may be incapable of T cell recognition because of a failure to interact with MHC restriction elements and/or a failure of MHC/carbohydrate complexes to interact with and be recognized by Ag-specific TCR. We have used two approaches to obtain information about T cell recognition of carbohydrate. First, we have determined the capacity of a series of oligosaccharides and glycolipids to bind a murine class II MHC molecule, IAd. No significant binding was observed with the 26 compounds tested, but the limitation to these studies was that there was a relatively limited collection of synthetic carbohydrate and glycolipid structures of limited complexity available for analysis. The second approach involved the study of the effect of glycosylation of a known peptide T cell epitope (OVA 323-339) on MHC binding of the peptide and on T cell recognition. Three patterns of effects were observed: 1) no effect on either binding or T cell recognition. This pattern was observed when the carbohydrate was located at residues removed from the core MHC-binding region. When the carbohydrate was located within the core MHC-binding regions, either 2) glycosylation destroyed both MHC binding and T cell recognition; or 3) glycosylation did not ablate MHC binding or T cell recognition. In this latter instance, there was evidence to indicate that the carbohydrate moiety was an important part of the antigenic determinant recognized by T cells.     

The C-4 hydroxyl group of galactopyranosides is the major determinant for ligand recognition by the lactose permease of Escherichia coli.

Binding specificity in lactose permease toward galactopyranosides is governed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while binding affinity can be increased dramatically by nonspecific hydrophobic interactions with the non-galactosyl moiety [Sahin-Tóth, M., Akhoon, K. M., Runner, J., and Kaback, H. R. (2000) Biochemistry 39, 5097-5103]. To characterize the contribution of individual hydroxyls, binding of structural analogues of p-nitrophenyl alpha-D-galactopyranoside (NPG) was examined by site-directed N-[(14)C]ethylmaleimide (NEM) labeling of the substrate-protectable Cys148 in the binding site. NPG blocks NEM alkylation of Cys148 with an apparent affinity of approximately 14 microM. A deoxy derivative at position C-2 binds with 25-fold lower affinity (K(D) 0.35 mM), and the deoxy analogue at C-3 exhibits ca. 70-fold decreased binding (K(D) 1 mM), while binding of 6-deoxy-NPG is at least 130-fold diminished (K(D) 1.9 mM). Remarkably, the C-4 deoxy derivative of NPG binds with almost 1500-fold reduced affinity (K(D) approximately 20 mM). No significant substrate protection is afforded by NPG analogues with methoxy (CH(3)-O-) substitutions at positions C-3, C-4, and C-6. In contrast, the C-2 methoxy analogue binds almost normally (K(D) 26 microM). The results confirm and extend the observations that the C-2, C-3, C-4, and C-6 OH groups of galactopyranosides participate in important H-bonding interactions. Moreover, the C-4 hydroxyl is identified as the major determinant of ligand binding, suggesting that sugar recognition in lactose permease may have evolved to discriminate primarily between gluco- and galactopyranosides.     

Metadynamics modelling of the solvent effect on primary hydroxyl rotamer equilibria in hexopyranosides

Accurate modelling of rotamer equilibria for the primary hydroxyl groups of monosaccharides continues to be a great challenge of computational glycochemistry. The metadynamics technique was applied to study the conformational free energy surfaces of methyl α-d-glucopyranoside and methyl α-d-galactopyranoside, employing the glycam06 force field. For both molecules, seven to eight conformational free-energy minima, differing in the ω (O-5–C-5–C-6–O-6) and χ (C-3–C-4–O-4–HO-4) dihedral angles, were identified in vacuum or in a water environment. The calculated rotamer equilibrium of the primary hydroxyl group is significantly different in vacuum than in water. The major effect of a water environment is the destabilisation of a hydrogen bond between O-4–HO-4 and O-6–HO-6 groups. It was possible to calculate the free-energy differences of individual rotamers with an accuracy of better than 2 kJ/mol. The calculated gg, gt and tg rotamer populations in water are in close agreement with experimental measurements, and therefore support the theoretical background of metadynamics.     

Range of the solvation pressure between lipid membranes: dependence on the packing density of solvent molecules

Well-ordered multilamellar arrays of liquid-crystalline phosphatidylcholine and equimolar phosphatidylcholine-cholesterol bilayers have been formed in the nonaqueous solvents formamide and 1,3-propanediol. The organization of these bilayers and the interactions between apposing bilayer surfaces have been investigated by X-ray diffraction analysis of liposomes compressed by applied osmotic pressures up to 6 X 10(7) dyn/cm2 (60 atm). The structure of egg phosphatidylcholine (EPC) bilayers in these solvents is quite different than in water, with the bilayer thickness being largest in water, 3 A narrower in formamide, and 6 A narrower in 1,3-propanediol. The incorporation of equimolar cholesterol increases the thickness of EPC bilayers immersed in each solvent, by over 10 A in the case of 1,3-propanediol. The osmotic pressures of various concentrations of the neutral polymer poly(vinylpyrrolidone) dissolved in formamide or 1,3-propanediol have been measured with a custom-built membrane osmometer. These measurements are used to obtain the distance dependence of the repulsive solvation pressure between apposing bilayer surfaces. For each solvent, the solvation pressure decreases exponentially with distance between bilayer surfaces. However, for both EPC and EPC-cholesterol bilayers, the decay length and magnitude of this repulsive pressure strongly depend on the solvent. The decay length for EPC bilayers in water, formamide, and 1,3-propanediol is found to be 1.7, 2.4, and 2.6 A, respectively, whereas the decay length for equimolar EPC-cholesterol bilayers in water, formamide, and 1,3-propanediol is found to be 2.1, 2.9, and 3.1 A, respectively. These data indicate that the decay length is inversely proportional to the cube root of the number of solvent molecules per unit volume.(ABSTRACT TRUNCATED AT 250 WORDS)     

环境胁迫对海草非结构性碳水化合物储存和转移的影响

非结构性碳水化合物在海草体内的代谢对植株的生长有重要影响。为更好地跟踪非结构性碳水化合物在海草响应环境胁迫中所起的作用,根据国内外最新文献,重点综述了光强、营养盐、盐度、海洋酸化、温度、硫化物和动物摄食等环境胁迫对海草非结构性碳水化合物储存和转移的影响。光限制和富营养化均降低非结构性碳水化合物的合成,并使之从地下根茎转移到叶;而海洋酸化却促进非结构性碳水化合物合成并向地下组织转移;盐度变化改变海草体内渗透压,需要非结构性碳水化合物的新陈代谢来维持;温度通过影响光合作用、呼吸作用、氮代谢来影响非结构性碳水化合物的合成与储存;而硫化物和动物摄食则分别通过抑制海草酶的活性和啃食海草光合组织,减少非结构性碳水化合物的合成和储存。同时指出了一些今后关于海草非结构性碳水化合物的重点研究方向:(1)海草不同生命阶段(种子休眠和萌发,发育,繁殖等)非结构性与结构性碳水化合物之间,以及可溶糖与淀粉之间的转化分配机制;(2)双环境因子或者多环境因子对海草非结构性碳水化合物的耦合作用;(3)非结构性碳水化合物作为海草床生态系统健康评价指标的研究与应用。     

Side-chain torsional potentials: effect of dipeptide, protein, and solvent environment.

Side-chain torsional potentials in the bovine pancreatic trypsin inhibitor are calculated from empirical energy functions by use of the known X-ray structure of the protein and the rigid-geometry mapping technique. The potentials are analyzed to determine the roles and relative importance of contributions from the dipeptide backbone, the protein, and the crystalline environment of solvent and other protein molecules. The structural characteristics of the side chains determine two major patterns of energy surfaces, E(X1,X2): a gamma-branched pattern and a pattern for longer, straight side chains (Arg, Lys, Glu, and Met). Most of the dipeptide potential curves and surfaces have a local minimum corresponding to the side-chain torsional angles in the X-ray structure. Addition of the protein forces sharpens and/or selects from these minima, providing very good agreement with the experimental conformation for most side chains at the surface or in the core of the protein. Inclusion of the crystalline environment produces still better results, especially for the side chains extending away from the protein. The results are discussed in terms of the details of the interactions due to the surrounding, calculated solvent-accessibility figures and the temperature factors derived from the crystallographic refinement of the pancreatic trypsin inhibitor.     

Cohesin-dockerin interaction in cellulosome assembly: a single hydroxyl group of a dockerin domain distinguishes between nonrecognition and high affinity recognition

The assembly of enzyme components into the cellulosome complex is dictated by the cohesin-dockerin interaction. In a recent article (Mechaly, A., Yaron, S., Lamed, R., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Shoham, Y., and Bayer, E. A. (2000) Proteins 39, 170-177), we provided experimental evidence that four previously predicted dockerin residues play a decisive role in the specificity of this high affinity interaction, although additional residues were also implicated. In the present communication, we examine further the contributing factors for the recognition of a dockerin by a cohesin domain between the respective cellulosomal systems of Clostridium thermocellum and Clostridium cellulolyticum. In this context, the four confirmed residues were analyzed for their individual effect on selectivity. In addition, other dockerin residues were discerned that could conceivably contribute to the interaction, and the suspected residues were similarly modified by site-directed mutagenesis. The results indicate that mutation of a single residue from threonine to leucine at a given position of the C. thermocellum dockerin differentiates between its nonrecognition and high affinity recognition (K(a) approximately 10(9) m(-1)) by a cohesin from C. cellulolyticum. This suggests that the presence or absence of a single decisive hydroxyl group is critical to the observed biorecognition. This study further implicates additional residues as secondary determinants in the specificity of interaction, because interconversion of selected residues reduced intraspecies self-recognition by at least three orders of magnitude. Nevertheless, as the latter mutageneses served to reduce but not annul the cohesin-dockerin interaction within this species, it follows that other subtle alterations play a comparatively minor role in the recognition between these two modules.