Recognition of selected monosaccharides by Pseudomonas aeruginosa Lectin II analyzed by molecular dynamics and free energy calculations

In this study, interactions of selected monosaccharides with the Pseudomonas aeruginosa Lectin II (PA-IIL) are analyzed in detail. An interesting feature of the PA-IIL binding is that the monosaccharide is interacting via two calcium ions and the binding is unusually strong for protein-saccharide interaction. We have used Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) and normal mode analysis to calculate the free energy of binding. The impact of intramolecular hydrogen bond network for the lectin/monosaccharide interaction is also analyzed.     

Preparation of DL-2,3,4-trihydroxybutylarsonic acid and dl-2,3-dihydroxybutane-1,4-bis(arsonic acid): starting compounds for novel arsonolipids

The reaction of DL-1,3-butadiene diepoxide and of DL-1,4-dibromo-2,3-butanediol with aqueous alkaline sodium arsenite, "Na(3)AsO(3)", gave mixtures of the title arsonic acids which can be separated by anion exchange resin. Characterization of by-products leads to a better understanding of these reactions. These compounds are valuable intermediates for the preparation of novel arsonic acids and bis(arsonic acids).     

Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins

D-amino acid amidase (DAA) from Ochrobactrum anthropi SV3, which catalyzes the stereospecific hydrolysis of D-amino acid amides to yield the D-amino acid and ammonia, has attracted increasing attention as a catalyst for the stereospecific production of D-amino acids. In order to clarify the structure-function relationships of DAA, the crystal structures of native DAA, and of the D-phenylalanine/DAA complex, were determined at 2.1 and at 2.4 A resolution, respectively. Both crystals contain six subunits (A-F) in the asymmetric unit. The fold of DAA is similar to that of the penicillin-recognizing proteins, especially D-alanyl-D-alanine-carboxypeptidase from Streptomyces R61, and class C beta-lactamase from Enterobacter cloacae strain GC1. The catalytic residues of DAA and the nucleophilic water molecule for deacylation were assigned based on these structures. DAA has a flexible Omega-loop, similar to class C beta-lactamase. DAA forms a pseudo acyl-enzyme intermediate between Ser60 O(gamma) and the carbonyl moiety of d-phenylalanine in subunits A, B, C, D, and E, but not in subunit F. The difference between subunit F and the other subunits (A, B, C, D and E) might be attributed to the order/disorder structure of the Omega-loop: the structure of this loop cannot assigned in subunit F. Deacylation of subunit F may be facilitated by the relative movement of deprotonated His307 toward Tyr149. His307 N(epsilon2) extracts the proton from Tyr149 O(eta), then Tyr149 O(eta) attacks a nucleophilic water molecule as a general base. Gln214 on the Omega-loop is essential for forming a network of water molecules that contains the nucleophilic water needed for deacylation. Although peptidase activity is found in almost all penicillin-recognizing proteins, DAA lacks peptidase activity. The lack of transpeptidase and carboxypeptidase activities may be attributed to steric hindrance of the substrate-binding pocket by a loop comprised of residues 278-290 and the Omega-loop.     

Protein composition analysis of oil bodies from maize embryos during germination

Seed oil bodies (OBs) are intracellular particles that store lipids. In maize embryos, the oil bodies are accumulated mainly in the scutellum. Oil bodies were purified from the scutellum of germinating maize seeds and the associated proteins were extracted and subjected to 2-DE analysis followed by LC-MS/MS for protein identification. In addition to the previously known oil body proteins oleosin, caleosin and steroleosin, new proteins were identified.     

H, C NMR and UV spectroscopy studies of gold(III)-tetracyanide complex with l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine in aqueous solution

Auricyanide [Au(CN)₄]⁻ interaction with biologically important thiols, thioether and selenoether were carried out and monitored using ¹H, ¹³C NMR and UV spectroscopy. These ligands include l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine. Thiols show very strong affinity to be oxidized into the disulfide by auricyanide, which gets reduced to aurocyanide [Au(CN)₂]⁻. l-cysteine reaction mechanism with [Au(CN)₄]⁻ was found to be dependent on reactants mole ratio. While l-methionine was completely inert toward auricyanide, dl-Se-methionine showed some reactivity with [Au(CN)₄]⁻ after raising solution pH to 12 that facilitated cyanide exchange.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Binding of different monosaccharides by lectin PA-IIL from Pseudomonas aeruginosa: thermodynamics data correlated with X-ray structures

The lectin from Pseudomonas aeruginosa (PA-IIL) is involved in host recognition and biofilm formation. Lectin not only displays an unusually high affinity for fucose but also binds to L-fucose, L-galactose and D-arabinose that differ only by the group at position 5 of the sugar ring. Isothermal calorimetry experiments provided precise determination of affinity for the three methyl-glycosides and revealed a large enthalpy contribution. The crystal structures of the complexes of PA-IIL with L-galactose and Met-beta-D-arabinoside have been determined and compared with the PA-IIL/fucose complex described previously. A combination of the structures and thermodynamics provided clues for the role of the hydrophobic group in affinity.     

Characterization of a GDP-D-mannose 3',5'-epimerase from rice

The enzymatic characterization of GDP-d-mannose 3',5'-epimerase (GME), a key enzyme in the biosynthesis of vitamin C in plants is described. The GME gene (Genbank Accession No. AB193582) in rice was cloned, and expressed as a fusion protein in Escherichia coli. Reaction products from GDP-d-mannose, as produced by GME catalysis, were separated by recycling HPLC on an ODS column, and were determined to be GDP-l-galactose and GDP-l-gulose, based on their NMR spectra and sugar analysis. The reaction catalyzed by GME was inhibited by GDP, and was strongly accelerated by NAD(+) in contrast to the case of GME from Arabidopsis thaliana. This difference in the effect of NAD(+) on GME activity can be attributed to the NAD binding domain which is conserved in the rice gene, but not in the Arabidopsis thaliana gene. The apparent K(m) and k(cat) were determined to be 1.20x10(-5)M and 0.127s(-1), respectively, in the presence of 20microM NAD(+). The fractions of GDP-d-mannose, GDP-l-galactose and GDP-l-gulose, at equilibrium, were approximately 0.75, 0.20 and 0.05, respectively.     

Crystal structure of YihS in complex with D-mannose: structural annotation of Escherichia coli and Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerase

The three-dimensional structure of a Salmonella enterica hypothetical protein YihS is significantly similar to that of N-acyl-d-glucosamine 2-epimerase (AGE) with respect to a common scaffold, an α₆/α₆-barrel, although the function of YihS remains to be clarified. To identify the function of YihS, Escherichia coli and S. enterica YihS proteins were overexpressed in E. coli, purified, and characterized. Both proteins were found to show no AGE activity but showed cofactor-independent aldose-ketose isomerase activity involved in the interconversion of monosaccharides, mannose, fructose, and glucose, or lyxose and xylulose. In order to clarify the structure/function relationship of YihS, we determined the crystal structure of S. enterica YihS mutant (H248A) in complex with a substrate (d-mannose) at 1.6 Å resolution. This enzyme-substrate complex structure is the first demonstration in the AGE structural family, and it enables us to identify active-site residues and postulate a reaction mechanism for YihS. The substrate, β-d-mannose, fits well in the active site and is specifically recognized by the enzyme. The substrate-binding site of YihS for the mannose C1 and O5 atoms is architecturally similar to those of mutarotases, suggesting that YihS adopts the pyranose ring-opening process by His383 and acidifies the C2 position, forming an aldehyde at the C1 position. In the isomerization step, His248 functions as a base catalyst responsible for transferring the proton from the C2 to C1 positions through a cis-enediol intermediate. On the other hand, in AGE, His248 is thought to abstract and re-adduct the proton at the C2 position of the substrate. These findings provide not only molecular insights into the YihS reaction mechanism but also useful information for the molecular design of novel carbohydrate-active enzymes with the common scaffold, α₆/α₆-barrel.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Purification and Characterization of a Magnesium Ion Requiring N-Acetyl-D-glucosamine Specific Lectin from Seeds of Quercus ilex L.

A new magnesium ion requiring N-acetyl-D-glucosamine specific lectin QIL was purified to electrophoretic homogeneity from seeds of Quercus ilex L. through successive steps of (i) lectin extraction, (ii) ammonium sulphate (30–50%) fractionation, (iii) diethylaminoethyl (DEAE)-cellulose chromatography, (iv) carboxymethyl (CM)-cellulose chromatography, and (v) Sephadex G-75 chromatography. The lectin, having specific activity of 25,600 hemagglutination units (HAU)/mg of protein, was found to be a monomeric protein with a native molecular weight of 13.2 kDa. N-Acetyl-D-glucosamine was found to exhibit most potent inhibitory action on the lectin activity among all the sugars tested. The lectin was also found to exhibit specificity for human blood groups A, B, and AB. It was converted to the corresponding apo-lectin by ethylenediaminetetraacetic acid (EDTA) treatment followed by buffer dialysis. The apo-lectin exhibited a specific and characteristic requirement for magnesium ions for the expression of its activity.     

Thermodynamic determination of the binding constants of angiotensin-converting enzyme inhibitors by a displacement method

Somatic angiotensin I-converting enzyme (s-ACE) plays a central role in blood pressure regulation and has been the target of most antihypertensive drugs. A displacement isothermal titration calorimetry method has been used to accurately determine the binding constant of three strong s-ACE inhibitors. Under the experimental conditions studied in this work, the relative potency of the inhibitors was determined to be enalaprilat>lisinopril>captopril. We analyze the thermodynamic behaviour of the binding process using the new structural information provided by the ACE structures, as well as the conformational changes that occur upon binding.     

Synthesis of 2,3,4,5-tetra-O-methyl-D-glucono-1,6-lactone as a monomer for the preparation of copolyesters

2,3,4,5-tetra-O-methyl-D-glucono-1,6-lactone has been prepared as a crystalline compound in acceptable yield by two different routes. An initial assay of copolymerization with L-lactide by ring-opening polymerization was carried out. The incorporation of the carbohydrate monomer into the polymer chain was about 2%.     

Molecular simulation of solid–solid phase transitions

Applications of dl_poly to solid–solid phase transitions are reviewed, with particular attention to how details of the mechanisms of the transitions may be extracted from molecular dynamics simulations. Two examples in molecular crystals are discussed: the order–disorder transition of p-terphenyl initiated at around 200 K by the unlocking of ring flipping; and the rotator phases of n-alkanes with around 20 carbon atoms per chain, showing distinct molecular mechanisms in the dynamics just below the melting points of odd and even chains. Covalent-ionic materials are represented by an application to an aluminophophate molecular sieve, AlPO₄-5.     

Characterization of human D-amino acid oxidase

D-Amino acid oxidase (DAAO) has been proposed to be involved in the oxidation of D-serine, an allosteric activator of the NMDA-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The recombinant human DAAO was expressed in Escherichia coli and was isolated as an active homodimeric flavoenzyme. It shows the properties of the dehydrogenase-oxidase class of flavoproteins, possesses a low kinetic efficiency, and follows a ternary complex (sequential) kinetic mechanism. In contrast to the other known DAAOs, the human enzyme is a stable homodimer even in the apoprotein form and weakly binds the cofactor in the free form.     

Structure of the O-polysaccharide of Proteus vulgaris O44: a new O-antigen that contains an amide of D-glucuronic acid with L-alanine

The O-polysaccharide of Proteus vulgaris O44, strain PrK 67/57 was studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H, 13C HMQC, HMQC-TOCSY and HMBC experiments. The polysaccharide was found to contain an amide of D-glucuronic acid with L-alanine [D-GlcA6(L-Ala)], and the following structure of the linear pentasaccharide repeating unit was established: [structure: see text]. The structural data of the O-polysaccharide and the results of serological studies with P. vulgaris O44 O-antiserum showed that the strain studied is unique among Proteus bacteria, which is in agreement with its classification in a separate Proteus serogroup, O44.     

A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.     

The effects of multiple ancestral residues on the Thermus thermophilus 3-isopropylmalate dehydrogenase

Previously, we showed that mutants of Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) each containing a residue (ancestral residue) that had been predicted to exist in a postulated common ancestor protein often have greater thermal stabilities than does the contemporary wild-type enzyme. In this study, the combined effects of multiple ancestral residues were analyzed. Two mutants, containing multiple mutations, Sup3mut (Val181Thr/Pro324Thr/Ala335Glu) and Sup4mut (Leu134Asn/Val181Thr/Pro324Thr/Ala335Glu) were constructed and show greater thermal stabilities than the wild-type and single-point mutant IPMDHs do. Most of the mutants have similar or improved catalytic efficiencies at 70 degrees C when compared with the wild-type IPMDH.     

Crystal Structure of Bacillus cereusd-Alanyl Carrier Protein Ligase (DltA) in Complex with ATP

d-Alanylation of lipoteichoic acids modulates the surface charge and ligand binding of the Gram-positive cell wall. Disruption of the bacterial dlt operon involved in teichoic acid alanylation, as well as inhibition of the DltA (d-alanyl carrier protein ligase) protein, has been shown to render the bacterium more susceptible to conventional antibiotics and host defense responses. The DltA catalyzes the adenylation and thiolation reactions of d-alanine. This enzyme belongs to a superfamily of AMP-forming domains such as the ubiquitous acetyl-coenzyme A synthetase. We have determined the 1.9-Å-resolution crystal structure of a DltA protein from Bacillus cereus in complex with ATP. This structure sheds light on the geometry of the bound ATP. The invariant catalytic residue Lys492 appears to be mobile, suggesting a molecular mechanism of catalysis for this superfamily of enzymes. Specific roles are also revealed for two other invariant residues: the divalent cation-stabilizing Glu298 and the β-phosphate-interacting Arg397. Mutant proteins with a glutamine substitution at position 298 or 397 are inactive.     

Synthesis of D- and L-myo-inositol 2,4,5-trisphosphate and trisphosphorothioate: structural analogues of D-myo-inositol 1,4,5-trisphosphate

The preparation of D- and L-myo-inositol 2,4,5-trisphosphate is described, together with the phosphorothioate counterparts. The known chiral diols D- and L-1,4-di-O-benzyl-5,6-bis-O-p-methoxybenzyl-myo-inositol were regioselectively protected at the 3-position using a benzyl group via a 2,3-O-stannylene acetal. Removal of the p-methoxybenzyl groups of each enantiomer gave D- and L-1,3,6-tri-O-benzyl-myo-inositol. Phosphitylation with bis(benzyloxy)diisoproplyaminophosphine and 1H-tetrazole gave the trisphosphite intermediate for each enantiomer. Oxidation with 3-chloroperoxybenzoic acid gave the fully protected D- and L-myo-inositol 2,4,5-trisphosphates. Sulphoxidation of the D- and L-2,4,5-trisphosphite intermediates gave the fully protected D- and L-myo-inositol 2,4,5-trisphosphorothioate compounds. The fully protected trisphosphates were deblocked using hydrogenolysis and the phosphorothioates were deprotected using sodium in liquid ammonia. The individual compounds were then purified using ion exchange chromatography to afford pure D- and L-myo-inositol 2,4,5-trisphosphates together with the corresponding phosphorothioates.