Suitable Acceptor Molecule Research Articles

Acceptor Specificity of Amylosucrase from Deinococcus radiopugnans and Its Application for Synthesis of Rutin Derivatives.

The transglycosylation activity of amylosucrase (ASase) has received significant attention owing to its use of an inexpensive donor, sucrose, and broad acceptor specificity, including glycone and aglycone compounds. The transglycosylation reaction of recombinant ASase from Deinococcus radiopugnans (DRpAS) was investigated using various phenolic compounds, and quercetin-3-O-rutinoside (rutin) was found to be the most suitable acceptor molecule used by DRpAS. Two amino acid residues in DRpAS variants (DRpAS Q299K and DRpAS Q299R), assumed to be involved in acceptor binding, were constructed by site-directed mutagenesis. Intriguingly, DRpAS Q299K and DRpAS Q299R produced 10-fold and 4-fold higher levels of rutin transglycosylation product than did the wild-type (WT) DRpAS, respectively. According to in silico molecular docking analysis, the lysine residue at position 299 in the mutants enables rutin to more easily position inside the active pocket of the mutant enzyme than in that of the WT, due to conformational changes in loop 4.

Fluorescence spectroscopy studies on polymer blend solutions and films for photovoltaics

Abstract The polymer TQ1, (poly[2,3-bis-(3-octyloxyphenyl)quinoxaline-5,8-diyl-alt-thiophene-2,5-diyl]) is a popular candidate as donor in organic solar cells. A suitable acceptor molecule together with TQ1 is [6,6]-phenyl-C71-butyric acid methyl ester (PC70BM). We have studied films of TQ1 by fluorescence spectroscopy, with and without PC70BM, in solution and spin-casted from different solvents. The solvents used in this study were chloroform, chlorobenzene, and ortho-dichlorobenzene, while 1-chloronaphthalene was used as solvent additive. From a spectroscopic point of view, TQ1 acts as fluorescent probe and PC70BM as a fluorescence quencher. Lateral phase-separation occurs in the blend films and its magnitude depends on the solvent used. These morphological differences cause alterations in the fluorescence from TQ1. The degree of quenching is coupled to the morphology through the distance between TQ1 and PC70BM in the film.

Tricin biosynthesis during growth of wheat under different abiotic stresses

In plants, O-methylation is mediated by an enzyme family of O-methyltransferases (OMTs) that transfer the methyl groups from the methyl donor, S-adenosyl-L-methionine (AdoMet) to suitable phenolic acceptor molecules. In a previous study [1], a flavonoid OMT (TaOMT2) was isolated and characterized from wheat (Triticum aestivum L.) leaves. Its novel gene product catalyzes three sequential O-methylations of the flavone tricetin (5,7,3′,4′,5′-pentahydroxyflavone) to its 3′-monomethyl-(selgin)→3′,5′-dimethyl-(tricin)→3′,4′,5′-trimethyl (TMT) ether derivatives, with tricin being the major product of the reaction. In this report, the biological significance of tricetin methylation was investigated by measuring the OMT activity, its expression level, and the accumulation of its major product (tricin) at different stages of development of wheat plants exposed to different abiotic stresses such as cold, salt and drought. The results showed that tricin accumulates mostly in wheat inflorescences under normal conditions compared to leaves and other developmental stages. Tricin accumulation was associated with increased TaOMT2 expression level and its enzyme activity, suggesting a possible de novo synthesis of the enzyme at this important developmental stage. This phenomenon may be attributed to the putative role of tricin in protecting seeds against biotic and abiotic stresses. The functions of tricin during growth and development of wheat and the importance of tricetin methylation during abiotic stresses are discussed.

Graphene-Based Chemiluminescence Resonance Energy Transfer for Homogeneous Immunoassay

We report on chemiluminescence resonance energy transfer (CRET) between graphene nanosheets and chemiluminescent donors. In contrast to fluorescence resonance energy transfer, CRET occurs via nonradiative dipole-dipole transfer of energy from a chemiluminescent donor to a suitable acceptor molecule without an external excitation source. We designed a graphene-based CRET platform for homogeneous immunoassay of C-reactive protein (CRP), a key marker for human inflammation and cardiovascular diseases, using a luminol/hydrogen peroxide chemiluminescence (CL) reaction catalyzed by horseradish peroxidase. According to our results, anti-CRP antibody conjugated to graphene nanosheets enabled the capture of CRP at the concentration above 1.6 ng mL(-1). In the CRET platform, graphene played a key role as an energy acceptor, which was more efficient than graphene oxide, while luminol served as a donor to graphene, triggering the CRET phenomenon between luminol and graphene. The graphene-based CRET platform was successfully applied to the detection of CRP in human serum samples in the range observed during acute inflammatory stress.

Sulfurtransferases 1 and 2 Play Essential Roles in Embryo and Seed Development in Arabidopsis thaliana

Sulfurtransferases (STRs) catalyze the transfer of a sulfur atom from a donor to a suitable acceptor molecule. The Arabidopsis thaliana genome encodes 20 putative STR proteins. The biological functions of most are unclear. We found that STR1 and STR2 play important roles in embryo/seed development. Mutation of STR1 alone resulted in a shrunken seed phenotype, although growth and development of vegetative and reproductive organs were not affected. The shrunken seed phenotype was associated with the delayed/arrested embryo development, in most cases, at the heart stage. The embryo defect of str1 mutant is not fully penetrant. Approximately 12.5% of embryos developed further and formed normal looking seeds. In severely shrunken seeds, no embryo could be identified after seed collection. Partially shrunken seeds that contained viable embryos could still germinate. However, cotyledons of the seedlings from such seeds were abnormal. An STR1-GUS fusion reporter revealed that the STR1 gene was universally expressed, with high levels of expression in specific tissues/organs including embryos. The incomplete penetrance of str1 embryo/seed phenotype is a result of functional STR2. Single str2 mutant had no phenotype. However, no str1(-/-)/str2(-/-) double mutant embryos were able to develop past the heart stage. Furthermore, STR2 is haplo-insufficient in str1 mutant background, and str1(-/-)/str2(+/-) embryos were 100% lethal. These data provide new insights into the biological functions of the ubiquitous sulfurtransferase in Arabidopsis embryogenesis and seed development.

A Bifunctional Enzyme for a Michael Addition-Oxidation Sequence

A biocatalytic hydration-oxidation sequence of α,β-unsaturated carbonyl compounds is reported. The enzyme, a Michael ­hydratase-alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans, enabled the efficient Michael addition of water to suitable acceptor molecules. Moreover, in the presence of a biocompatible electron acceptor, such as methylene blue or dichlorophenol indophenol, the oxidation to the dicarbonyl compound could also be catalyzed by the same enzyme. The biocatalytic system tolerated aldehydes and ketones equally well. The enantioselectivity of the enzyme could be demonstrated, as upon incubation of the biocatalyst with an enantiomerically pure (R) versus the racemic alcohol substrate the former was oxidized at higher rate.

Sucrose phosphorylase: a powerful transglucosylation catalyst for synthesis of α-D-glucosides as industrial fine chemicals

Sucrose phosphorylase is a bacterial transglucosidase that catalyzes conversion of sucrose and phosphate into α-D-glucose-1-phosphate and D-fructose. The enzyme utilizes a glycoside hydrolase-like double displacement mechanism that involves a catalytically competent β-glucosyl enzyme intermediate. In addition to reaction with phosphate, glucosylated sucrose phosphorylase can undergo hydrolysis to yield α-D-glucose or it can decompose via glucosyl transfer to a hydroxy group in suitable acceptor molecules, giving new α-D-glucosidic products. The glucosyl acceptor specificity of sucrose phosphorylase is reviewed, focusing on applications of the enzyme in glucoside synthesis. Polyhydroxylated compounds such as sugars and sugar alcohols are often glucosylated efficiently. Aryl alcohols and different carboxylic acids also serve as acceptors for enzymatic transglucosylation. The natural osmolyte 2-O-(α-D-glucopyranosyl)-sn-glycerol (GG) was prepared by regioselective glucosylation of glycerol from sucrose using the phosphorylase from Leuconostoc mesenteroides. An industrial process for production of GG as active ingredient of cosmetic formulations has been recently developed. General advantages of sucrose phosphorylase as a transglucosylation catalyst lie in the use of sucrose as a high-energy glucosyl donor and the usually weak hydrolase activity of the enzyme towards substrate and product.

Acceptor products of alternansucrase with gentiobiose. Production of novel oligosaccharides for food and feed and elimination of bitterness

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. Previously, we showed that alternansucrase only forms certain isomers of DP > 4 from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. We now demonstrate that the acceptor products from gentiobiose, also formed in good yields (nearly 90% in unoptimized reactions), follow a pattern similar to those formed from maltose. The initial product is a single trisaccharide, α- d-Glc p-(1→6)-β- d-Glc p-(1→6)- d-Glc. Two tetrasaccharides were formed in approximately equal quantities: α- d-Glc p-(1→3)-α- d-Glc p-(1→6)-β- d-Glc p-(1→6)- d-Glc and α- d-Glc p-(1→6)-α- d-Glc p-(1→6)-β- d-Glc p-(1→6)- d-Glc. Just one pentasaccharide was isolated from the reaction mixture, α- d-Glc p-(1→6)-α- d-Glc p-(1→3)-α- d-Glc p-(1→6)-β- d-Glc p-(1→6)- d-Glc. Our hypothesis that the enzyme is incapable of forming two consecutive α-(1→3) linkages, and does not form products with more than two consecutive α-(1→6) linkages, apparently applies to other acceptors as well as to maltose. The glucosylation of gentiobiose reduces or eliminates its bitter taste.

Characterization of a novel bacterial arginine kinase from Desulfotalea psychrophila

Phosphagen kinases are found throughout the animal kingdom and catalyze the transfer of a high-energy gamma phosphoryl-group from ATP to a guanidino group on a suitable acceptor molecule such as creatine or arginine. Recent genome sequencing efforts in several proteobacteria, including Desulfotalea psychrophila LSv54, Myxococcus xanthus, Sulfurovum sp. NBC37-1, and Moritella sp. PE36 have revealed what appears to be a phosphagen kinase homolog present in their genomes. Based on sequence comparisons these putative homologs bear a strong resemblance to arginine kinases found in many invertebrates and some protozoa. We describe here a biochemical characterization of one of these homologs from D. psychrophila expressed in E. coli that confirms its ability to reversibly catalyze phosphoryl transfer from ATP to arginine. A phylogenetic analysis suggests that these bacteria homologs are not widely distributed in proteobacteria species. They appear more related to protozoan arginine kinases than to similar proteins seen in some Gram-positive bacteria that share key catalytic residues but encode protein tyrosine kinases. This raises the possibility of horizontal gene transfer as a likely origin of the bacterial arginine kinases.

Biochemical and crystallographic characterization of a glucansucrase from Lactobacillus reuteri 180

Glucansucrases are large extracellular transglycosidases secreted by lactic acid bacteria. Using sucrose as a substrate they synthesize high molecular mass α-glucans or, in the presence of suitable acceptor molecules, low molecular mass oligosaccharides. Although about 60 glucansucrases have been classified in glycoside hydrolase family GH70, no three-dimensional structure has been reported for any. With the aim of solving the first structure of a GH70 glucansucrase, purification and crystallization experiments were performed with a fully active, 117 kDa N-terminally truncated fragment of glucansucrase GTF180 from Lactobacillus reuteri 180 (residues 742–1772). Crystallization experiments yielded crystals that belong to two different triclinic crystal forms (space group P1) and one orthorhombic crystal form (space group P212121). Native data sets for both triclinic and the orthorhombic crystals were collected at 1.7 and 2.0 Å resolution, respectively. Enzyme activity assays, pH and temperature optima show comparable values for both the full-length and the N-terminally truncated GTF180.

Penta-, hexa-, and heptasaccharide acceptor products of alternansucrase

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. For the example of maltose as the acceptor, if one considers only the linear, unbranched possibilities for alternansucrase, the hypothetical number of potential products increases exponentially as a function of the degree of polymerization (DP). Experimental evidence indicates that far fewer products are actually formed. We show that only certain isomers of DP >4 are formed from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. When the oligosaccharide acceptor products from maltose were separated by size-exclusion chromatography and HPLC, only one pentasaccharide was isolated. Its structure was α-d-Glcp-(1→6)-α-d-Glcp-(1→3)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-d-Glc. Two hexasaccharides were formed in approximately equal quantities: α-d-Glcp-(1→3)-α-d-Glcp-(1→6)-α-d-Glcp-(1→3)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-d-Glc and α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→3)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-d-Glc. Just one heptasaccharide was isolated from the reaction mixture, α-d-Glcp-(1→6)-α-d-Glcp-(1→3)-α-d-Glcp-(1→6)-α-d-Glcp-(1→3)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-d-Glc. We conclude that the enzyme is incapable of forming two consecutive α-(1→3) linkages, and does not form products with more than two consecutive α-(1→6) linkages. The distribution of products may be kinetically determined.

Energy and Electron Transfers in Photosensitive Chitosan

Novel photosensitive chitosan was synthesized. The modified chitosan contains photoactive anthracene chromophore moieties. Because of the presence of anthracene chromophores, the polymer absorbs light in the UV-vis spectral region. Electronically excited polymeric chromophores could participate in energy and electron transfer processes to the suitable acceptor molecules. The photosensitive chitosan developed herein could could act as an efficient photosensitizer and lead to the application of the environmentally friendly photocatalytic system for an efficient degradation of a wide range of pollutants.

Development of a Time-Resolved Fluorometric Detection System Using Diffusion-Enhanced Energy Transfer

A novel detection system using both emission energy transfer and time-resolved fluorometry (TRF) was developed, with a europium chelate as the energy donor and a novel fluorophore SNR1, excitable with long-wavelength light corresponding to europium emission, as the energy acceptor. When the donor and acceptor molecules were mixed in solution, energy transfer was observed without direct attachment of the donor and the acceptor, via a diffusion-enhanced energy-transfer mechanism. Thus, the acceptor emission can be detected as a long-lifetime fluorescence in TRF. When the fluorescence properties of the acceptor molecule are changed by interaction with an enzyme or other bioactive molecule, the change can be detected as a long-lived sensitized emission. If we develop or select suitable acceptor molecules, this simple and convenient system should be applicable to a wide variety of bioactive molecules. Since it is based on TRF, it can be used for high-resolution assay.

Novel electron transfer polymerization processes with alkali metal supramolecular complex as catalyst

AbstractSingle electron transfer SET induced usually by organometallic complexes of transition metals or alkali metal complexes of arenes e.g. naphthalene is common in organic reactions. In this paper unusual two‐electron transfer to suitable acceptor molecule mediated by recently discovered alkali metal supramolecular complexes in aprotic etheral solvents is presented. The capability of these solutions of transferring two electrons to a suitable organic substrate and formation of corresponding carbanions opens new door in synthetic chemistry. The carbanions are interesting synthons in the many organic reactions and catalysts in anionic polymerization processes. Reactions of some vinyl compounds, alkyl and silyl halides, as well as ring‐opening polymerization of β‐lactones and oxiranes are discussed.

Hydrolase-catalyzed beta-mannosylations.

Transmannosylations catalysed by beta-mannosidase from snail viscera or beta-galactosidase from Aspergillus oryzae were accomplished with 4-nitrophenyl beta-D-mannopyranoside as donor substrate. With suitable hydrophobic acceptor molecules preferentially beta1-4-linked disaccharides were obtained. The activities of both glycosidases in buffer cosolvent mixtures were determined, and conditions for their immobilization were elaborated and optimized. A model of the enzymic transfer mechanism is suggested.

Cyclodextrins Are Not the Major Cyclic α-1,4-Glucans Produced by the Initial Action of Cyclodextrin Glucanotransferase on Amylose

The initial action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkalophilic Bacillus sp. A2-5a on amylose was investigated. Synthetic amylose was incubated with purified CGTase then terminated in the very early stage of the enzyme reaction. When the reaction mixture was treated with glucoamylase and the resulting glucoamylase-resistant glucans were analyzed with high performance anion exchange chromatography, cyclic alpha-1,4-glucans, with degree of polymerization ranging from 9 to more than 60, in addition to well known alpha-, beta-, and gamma-cyclodextrin (CD), were detected. The time-course analysis revealed that larger cyclic alpha-1, 4-glucans were preferentially produced in the initial stage of the cyclization reaction and were subsequently converted into smaller cyclic alpha-1,4-glucans and into the final major product, beta-CD. CGTase from Bacillus macerans also produced large cyclic alpha-1, 4-glucans except that the final major product was alpha-CD. Based on these results, a new model for the action of CGTase on amylose was proposed, which may contradict the widely held view of the cyclization reaction of CGTase.

Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi.

The gene(s) encoding the Trypanosoma cruzi shed-acute-phase-antigen (SAPA) has a 5' end encoding a region containing two totally and two partially conserved Ser-X-Asp-X-Gly-X-Thr-Trp motifs which are present in bacterial neuraminidases, and a 3' end encoding tandemly repeated units of 12 amino acids. It is now reported that 54-87% of the total neuraminidase activity present in the parasite could be immunoprecipitated with polyclonal or monoclonal antibodies against the repeated amino acid units of SAPA. These immunoprecipitates also had greater than 80% of the trans-sialidase activity of the parasite. SAPA used sialyllactose, fetuin and 4-methylumbelliferyl-sialic acid as substrate donors. In the presence of a suitable acceptor molecule (lactose) the sialic acid residues were transferred to the disaccharide, whereas in the absence of acceptors the residues were transferred to water. If relatively inefficient acceptors (maltose or cellobiose) were added to the incubation mixtures, the sialic acid units were transferred both to the disaccharides and to water. It is concluded that a major T. cruzi antigen has both the trans-sialidase and the neuraminidase activities of the parasite. Both activities are probably located on the N-terminus of SAPA since antibodies directed against the C-terminus, which contains the repeated amino acid units, do not affect the enzymatic activities.

Copper-induced dealkylation studies of biologically N-alkylated porphyrins by fast atom bombardment mass spectrometry

Copper has been reported to promote the demethylation of N-methylporphyrins. Copper-induced dealkylation is observed at room temperature in acetonitrile in the presence of a suitable nucleophilic acceptor molecule, or in chloroform alone. Electronic absorption spectra of N-methylprotoporphyrin IX (in the form of its dimethyl ester) in the presence of copper (with either chloroform alone or acetonitrile-dibutylamine as solvent) exhibited a transient spectrum corresponding to a copper chelate of the N-methylprotoporphyrin followed by ejection of the N-methyl group to afford the copper chelate of protoporphyrin. The use of fast atom bombardment mass spectrometry (FAB-MS) to identify the N-alkyl group of a series of N-substituted porphyrins was investigated. The alkyl group released on reaction with copper ions was trapped by an amine (e.g., dodecylamine). The resulting mixture was analysed by positive-ion FAB-MS using 3-nitrobenzyl alcohol as matrix. Molecular ions (MH +) were detected at m/z 200, 214 and 228, corresponding to [CH 3(CH 2) 11NHCH 3 + H] +, [CH 3(CH 2) 11NHC 2H 5 + H] + and [CH 3(CH 2) 11NHC 3H 7 + H] +, respectively. The analysis was extended to N-alkylprotoporphyrins isolated from liver homogenates of mice treated with the porphyria-inducing drug griseofulvin. Using a combination of UV and copper-induced dealkylation studies in FAB-MS, the porphyria-producing pigment was identified as N-methylprotoporphyrin IX.

CHARGE TRANSFER REACTIONS OF TRIVALENT PHOSPHORUS COMPOUNDS WITHP-QUINONES

Abstract Reactions of trivalent phosphorus compounds with suitable aromatic acceptor molecules proceed via a radical mechanism. In the case of p-quinones, the products and intermediates are identified by esr and an appropriate mechanism is outlined. The esr-data of the radicals are interpreted by means of spin density calculations.

γ-Glutamyl transpeptidase from kidney bean fruit I. Purification and mechanism of action

1. 1.|A γ-glutamyl transpeptidase from kidney bean fruit has been further purified and its properties have been studied. The purified material seems to consist of one component, as judged by its behavior in the ultracentrifuge and its elution pattern from an acrylamide-gel column. The preparation catalyzes the transfer of the γ-glutamyl moiety of glutathione, γ-glutamylaniline, and γ- glutamyl-p- nitroaniline to suitable acceptor molecules, usually amino acids. 2. 2.|The same preparation was found to catalyze the hydrolytic cleavage of the γ-glutamyl peptide linkage of these compounds. Evidence, based on purification, gel filtration and activation, is presented that the two activities are associated with the same enzyme. 3. 3.|From kinetic studies, it is concluded that a basic nitrogen of an imidazole group and a phenolic group of tyrosine constitute a part of the catalytic site of the enzyme molecule. On the basis of these findings a mechanism for the transfer and the hydrolytic reactions is proposed based on a nucleophilic attack of imidazole nitrogen on substrates. 4. 4.|It is suggested that the enzyme is involved in the degradation of glutathione.