Crystal structures of rare disaccharides, α-D-glucopyranosyl β-D-psicofuranoside, and α-D-galactopyranosyl β-D-psicofuranoside

The crystal structures of α-D-glucopyranosyl β-D-psicofuranoside and α-D-galactopyranosyl β-D-psicofuranoside were determined by a single-crystal X-ray diffraction analysis, refined to R(1)=0.0307 and 0.0438, respectively. Both disaccharides have a similar molecular structure, in which psicofuranose rings adopt an intermediate form between (4)E and (4)T(3). Unique molecular packing of the disaccharides was found in crystals, with the molecules forming a layered structure stacked along the y-axis.     

Synthesis and α-glucosidase inhibitory activity evaluation of N-substituted aminomethyl-β-d-glucopyranosides

A series of N-substituted 1-aminomethyl-β-d-glucopyranoside derivatives was prepared. These novel synthetic compounds were assessed in vitro for inhibitory activity against yeast α-glucosidase and both rat intestinal α-glucosidases maltase and sucrase. Most of the compounds displayed α-glucosidase inhibitory activity, with IC₅₀ values covering the wide range from 2.3 μM to 2.0 mM. Compounds 19a (IC₅₀ = 2.3 μM) and 19b (IC₅₀ = 5.6 μM) were identified as the most potent inhibitors for yeast α-glucosidase, while compounds 16 (IC₅₀ = 7.7 and 15.6 μM) and 19e (IC₅₀ = 5.1 and 10.4 μM) were the strongest inhibitors of rat intestinal maltase and sucrase. Analysis of the kinetics of enzyme inhibition indicated that 19e inhibited maltase and sucrase in a competitive manner. The results suggest that the aminomethyl-β-d-glucopyranoside moiety can mimic the substrates of α-glucosidase in the enzyme catalytic site, leading to competitive enzyme inhibition. Moreover, the nature of the N-substituent has considerable influence on inhibitory potency.     

Crystal structures of glycoside hydrolase family 51 α-L-arabinofuranosidase from Thermotoga maritima

α-L-Arabinofuranosidase from the hyperthermophilic bacterium Thermotoga maritima (Tm-AFase) is an extremely thermophilic enzyme belonging to glycoside hydrolase family 51. It can catalyze the transglycosylation of a novel glycosyl donor, 4,6-dimethoxy-1,3,5-triazin-2-yl (DMT)-β-D-xylopyranoside. In this study we determined the crystal structures of Tm-AFase in substrate-free and complex forms with arabinose and xylose at 1.8-2.3 ? resolution to determine the architecture of the substrate binding pocket. Subsite -1 of Tm-AFase is similar to that of α-L-arabinofuranosidase from Geobacillus stearothermophilus, but the substrate binding pocket of Tm-AFase is narrower and more hydrophobic. Possible substrate binding modes were investigated by automated docking analysis.     

The crystal and molecular structure of α-laminaribiose octa-acetate

The crystal and molecular structure of octa-O-acetyl-α-laminaribiose is compared with those of disaccharides, and their acetylated derivatives, related to polysaccharides of the laminarin and curdlan type. Marked differences are found in the endo- and exo-cyclic torsion angles as well as in the molecular geometry of the glycosidic linkage. The conformation of AcO-6′ provides the first experimental evidence of the theoretically predicted gauche-trans-gauche⁻ conformation. This conformational change, together with the α-orientation of AcO-1, alters the overall shape of the molecule and influences the packing features. The effect of the acetylation is also examined in terms of calculated conformational energy maps.     

Crystal and molecular structure of methyl L-glycero-α-D-manno-heptopyranoside, and synthesis of 1→7 linked L-glycero-D-manno-heptobiose and its methyl α-glycoside

Methyl l-glycero-α-d-manno-heptopyranoside was synthesized in good yield by a Fischer-type glycosylation of the heptopyranose with methanol in the presence of cation-exchange resin under reflux and microwave conditions, respectively. The compound crystallized from 2-propanol in an orthorhombic lattice of space group P2(1)2(1)2 showing a comparatively porous structure with a 2-dimensional O-H?O hydrogen bond network. As model compounds for the side chain domains of the inner core structure of bacterial lipopolysaccharide, l-glycero-α-d-manno-heptopyranosyl-(1→7)-l-glycero-d-manno-heptopyranose and the corresponding disaccharide methyl α-glycoside were prepared. The former compound was generated via glycosylation of a benzyl 5,6-dideoxy-hept-5-enofuranoside intermediate followed by catalytic osmylation and deprotection. The latter disaccharide was efficiently synthesized in good yield by a straightforward coupling of an acetylated N-phenyltrifluoroacetimidate heptopyranosyl donor to a methyl 2,3,4,6-tetra-O-acetyl heptopyranoside acceptor derivative followed by Zemplén deacetylation.     

A critical evaluation of the predicted and X-ray structures of α-Lactalbumin

The rapidly increasing availability of protein amino-acid sequences, many of which have been determined from the corresponding gene sequences, has intensified interest in the prediction of related protein structures when the three-dimensional structure of another member of the family is known. The study of bovine α-Lactalbumin provides a classic example in which the three-dimensional structure was predicted, first by Browneet al. (1969) and later by Warmeet al. (1974), from the three-dimensional structure of hen-egg-white lysozyme (Blakeet al., 1965), taking into account the striking relationship between the amino acid sequences of the two proteins. A comprehensive comparison of these models with the structure of baboon α-Lactalbumin derived from X-ray crystallography (Acharyaet al., 1989) is presented. The models mostly compare well with the experimentally determined structure except in the flexible C-terminal region of the molecule (rms deviation on C^α of residues 1–95, 1.1 Å).     

Frequency and molecular types of deletional α-thalassemia in Egypt

Summary The frequency of deletional -thalassemia in the Egyptian population was estimated at 0.08 by DNA analysis of a newborn random sample. No ⁰ determinants were found. The most frequent ⁺ determinant was the –^3.7 type I in association with the medium allele at inter-zeta HVR. The –^4.2 and ^{anti 3.7} arrangements were found at very low frequencies.     

Biotechnology and molecular biology of the α-glucosidase inhibitor acarbose

The -glucosidase inhibitor acarbose, O-{4,6-dideoxy-4[1s-(1,4,6/5)-4,5,6-trihydroxy-3-hydroxymethyl-2-cyclohexen-1-yl]-amino--d-glucopyranosyl}-(14)-O--d-glucopyranosyl-(14)-d-glucopyranose, is produced in large-scale fermentation by the use of strains derived from Actinoplanes sp. SE50. It has been used since 1990 in many countries in the therapy of diabetes type II, in order to enable patients to better control blood sugar contents while living with starch-containing diets. Thus, it is one of the latest successful products of bacterial secondary metabolism to be introduced into the pharmaceutical world market. Cultures of Actinoplanes sp. also produce various other acarbose-like components, of which component C is hard to separate during downstream processing, which is one of the most modern work-up processes developed to date. The physiology, genetics and enzymology of acarbose biosynthesis and metabolism in the producer have been studied to some extent, leading to the proposal of a new pathway and metabolic cycle, the carbophore. These data could give clues for further biotechnological developments, such as the suppression of side-products, enzymological or biocombinatorial production of new metabolites and the engineering of production rates via genetic regulation in future.     

Biochemical and molecular characterization of secreted α-xylosidase from Aspergillus niger

α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-d-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with β-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, β-glucosidase, xyloglucanase, and β-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi.     

Structural elucidation and molecular characterization of Marinobacter sp. α-amylase

Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Marinobacter sp. EMB8 α-amylase was found to be active and stable in salt and organic solvents. A study was carried out using circular dichroism (CD), fluorescence spectroscopy, and bioinformatics analysis of similar protein sequence to ascertain molecular basis of salt and solvent adaptability of α-amylase. Structural changes recorded in the presence of varying amounts of NaCl exhibited an increase in negative ellipticity as a function of salt, confirming that salt stabilizes the protein and increases the secondary structure, making it catalytically functional. The data of intrinsic and extrinsic fluorescence (using 1-anilinonaphthalene 8-sulfonate [ANS] as probe) further confirmed the role of salt. The α-amylase was active in the presence of nonpolar solvents, namely, hexane and decane, but inactivated by ethanol. The decrease in the activity was correlated with the loss of tertiary structure in the presence of ethanol. Guanidine hydrochloride and pH denaturation indicated the molten globule state at pH 4.0. Partial N-terminal amino acid sequence of the purified α-amylase revealed the relatedness to Pseudoalteromonas sp. α-amylase. “FVHLFEW” was found as the N-terminal signature sequence. Bioinformatics analysis was done using M. algicola α-amylase protein having the same N-terminal signature sequence. The three-dimensional structure of Marinobacter α-amylase was deduced using the I-TASSER server, which reflected the enrichment of acidic amino acids on the surface, imparting the stability in the presence of salt. Our study clearly indicate that salt is necessary for maintaining the secondary and tertiary structure of halophilic protein, which is a necessary prerequisite for catalysis.     

Enzymic synthesis and hydrolytic resolution of alkyl β-d-glucopyranosides

Extracellular -glucosidases from Aspergillus oryzae (two strains), Fusarium oxysporum, Aspergillus terreus and Talaromyces flavus were either induced by cellobiose or 6-deoxyglucose, or produced without induction, i.e., either on casein acid hydrolyzate or on the Sabouraud medium. They were tested to catalyze a stereoselective synthesis of the cis- and trans-isomers of 2-(4-methoxybenzyl)-1-cyclohexyl -d-glucopyranosides with either d-glucose or phenyl -d-glucopyranoside as sources of the glucosyl unit. The enzymes also hydrolyzed (resolved) diastereoisomeric mixtures of alkyl -d-glucopyranosides, which were prepared by the Koenigs–Knorr method from the respective cis- and trans-isomers of 2-(4-methoxybenzyl)-1-cyclohexanol.     

Energetics and structure of alanine-rich α-helices via adaptive steered molecular dynamics

The energetics and hydrogen bonding profiles of the helix-to-coil transition were found to be an additive property and to increase linearly with chain length, respectively, in alanine-rich α-helical peptides. A model system of polyalanine repeats was used to establish this hypothesis for the energetic trends and hydrogen bonding profiles. Numerical measurements of a synthesized polypeptide Ac-Y(AEAAKA)_kF-NH₂ and a natural α-helical peptide a2N (1–17) provide evidence of the hypothesis’s generality. Adaptive steered molecular dynamics was employed to investigate the mechanical unfolding of all of these alanine-rich polypeptides. We found that the helix-to-coil transition is primarily dependent on the breaking of the intramolecular backbone hydrogen bonds and independent of specific side-chain interactions and chain length. The mechanical unfolding of the α-helical peptides results in a turnover mechanism in which a 3₁₀-helical structure forms during the unfolding, remaining at a near constant population and thereby maintaining additivity in the free energy. The intermediate partially unfolded structures exhibited polyproline II helical structure as previously seen by others. In summary, we found that the average force required to pull alanine-rich α-helical peptides in between the endpoints—namely the native structure and free coil—is nearly independent of the length or the specific primary structure.     

Identification and molecular characterization of the mammalian α-kleisin RAD21L

Meiosis is a fundamental process that generates new combinations between maternal and paternal genomes and haploid gametes from diploid progenitors. Many of the meiosis-specific events stem from the behavior of the cohesin complex (CC), a proteinaceous ring structure that entraps sister chromatids until the onset of anaphase. CCs ensure chromosome segregation, participate in DNA repair, regulate gene expression, and also contribute to synaptonemal complex (SC) formation at meiosis by keeping long-range distant DNA interactions through its conserved structure. Studies from yeast to humans have led to the assumption that Scc1/RAD21 is the α-kleisin that closes the tripartite CC that entraps two DNA molecules in mitosis, while its paralog REC8 is essential for meiosis. Here we describe the identification of RAD21L, a novel mammalian CC subunit with homology to the RAD21/REC8 α-kleisin subfamily, which is expressed in mouse testis. RAD21L interacts with other cohesin subunits such as SMC1α, SMC1b, SMC3 and with the meiosis-specific STAG3 protein. Thus, our results demonstrate the existence of a new meiotic-specific CC constituted by this α-kleisin and expand the view of REC8 as the only specific meiotic α-kleisin.     

Crystal structures of human Ero1�� reveal the mechanisms of regulated and targeted oxidation of PDI

In the endoplasmic reticulum (ER) of eukaryotic cells, Ero1 flavoenzymes promote oxidative protein folding through protein disulphide isomerase (PDI), generating reactive oxygen species (hydrogen peroxide) as byproducts. Therefore, Ero1 activity must be strictly regulated to avoid futile oxidation cycles in the ER. Although regulatory mechanisms restraining Ero1α activity ensure that not all PDIs are oxidized, its specificity towards PDI could allow other resident oxidoreductases to remain reduced and competent to carry out isomerization and reduction of protein substrates. In this study, crystal structures of human Ero1α were solved in its hyperactive and inactive forms. Our findings reveal that human Ero1α modulates its oxidative activity by properly positioning regulatory cysteines within an intrinsically flexible loop, and by fine‐tuning the electron shuttle ability of the loop through disulphide rearrangements. Specific PDI targeting is guaranteed by electrostatic and hydrophobic interactions of Ero1α with the PDI b′‐domain through its substrate‐binding pocket. These results reveal the molecular basis of the regulation and specificity of protein disulphide formation in human cells.     

Crystal structure and thermostability of a putative α-glucosidase from Thermotoga neapolitana

Glycoside hydrolase family 4 (GH4) represents an unusual group of glucosidases with a requirement for NAD(+), Mn(2+), and reducing conditions. We found a putative α-glucosidase belonging to GH4 in hyperthermophilic Gram-negative bacterium Thermotoga neapolitana. In this study, we recombinantly expressed the putative α-glycosidase from T. neapolitana, and determined the crystal structure of the protein at a resolution of 2.0? in the presence of Mn(2+) but in the absence of NAD(+). The structure showed the dimeric assembly and the Mn(2+) coordination that other GH4 enzymes share. In comparison, we observed structural changes in T. neapolitana α-glucosidase by the binding of NAD(+), which also increased the thermostability. Numerous arginine-mediated salt-bridges were observed in the structure, and we confirmed that the salt bridges correlated with the thermostability of the proteins. Disruption of the salt bridge that linked N-terminal and C-terminal parts at the surface dramatically decreased the thermostability. A mutation that changed the internal salt bridge to a hydrogen bond also decreased the thermostability of the protein. This study will help us to understand the function of the putative glucosidase and the structural features that affect the thermostability of the protein.     

Syntheses, structures and luminescence of Europium α-thiophene carboxylates coordination polymer and supramolecular compound

Two europium α-thiophene carboxylic acid (HTPA) compounds, coordination polymer Eu(TPA)₃(HTPA)₂ (1) (TPA=α-thiophene carboxylate) and supramolecular compound Eu(TPA)₃(H₂O)₃ · 0.5H₂O (2) with luminescence and triboluminescence, have been synthesized and structurally characterized. In 1 each europium is bridged by six oxygen atoms from six carboxylates and coordinated with two carboxyl oxygen atoms from two α-thiophene carboxylic acid molecules, resulting in a coordination number of eight to Eu. For 2 each europium is chelated by six oxygen atoms from six carboxylates and coordinated with three oxygen atoms from three coordinated water generating a coordination number nine to Eu; A supramolecular compound is constructed through hydrogen bonds. Both 1 and 2 display strong characteristic emission of Eu³⁺ ion radiated by UV light and produce twinkling red light with an external force.     

Studies on molecular evolution and structural features of double-headed inhibitors of α-amylase and trypsin in plants

Plant seeds usually have high concentrations of proteinase and amylase inhibitors. These inhibitors exhibit a wide range of specificity, stability and oligomeric structure. In this communication, we report analysis of sequences that show statistically significant similarity to the double-headed α-amylase/trypsin inhibitor of ragi (Eleusine coracana). Our aim is to understand their evolutionary and structural features. The 14 sequences of this family that are available in the SWISSPROT database form three evolutionarily distinct branches. The branches relate to enzyme specificities and also probably to the oligomeric state of the proteins and not to the botanical class of the plant from which the enzymes are derived. This suggests that the enzyme specificities of the inhibitors evolved before the divergence of commercially cultivated cereals. The inhibitor sequences have three regions that display periodicity in hydrophobicity. It is likely that this feature reflects extended secondary structure in these segments. One of the most variable regions of the polypeptide corresponds to a loop, which is most probably exposed in the native structure of the inhibitors and is responsible for the inhibitory property.