Analysis Of Reaction Products Research Articles

Analysis of Reaction Products of Food Contaminants and Ingredients: Bisphenol A Diglycidyl Ether (BADGE) in Canned Foods

Bisphenol A diglycidyl ether (BADGE) is an epoxide that is used as a starting substance in the manufacture of can coatings for food-contact applications. Following migration from the can coating into food, BADGE levels decay and new reaction products are formed by reaction with food ingredients. The significant decay of BADGE was demonstrated by liquid chromatographic (LC) analysis of foodstuffs, that is, tuna, apple puree, and beer, spiked with BADGE before processing and storage. Life-science inspired analytical approaches were successfully applied to study the reactions of BADGE with food ingredients, for example, amino acids and sugars. An improved mass balance of BADGE was achieved by selective detection of reaction products of BADGE with low molecular weight food components, using a successful combination of stable isotopes of BADGE and analysis by LC coupled to fluorescence detection (FLD) and high-resolution mass spectrometric (MS) detection. Furthermore, proteomics approaches showed that BADGE also reacts with peptides (from protein digests in model systems) and with proteins in foods. The predominant reaction center for amino acids, peptides, and proteins was cysteine.

Isolation, Expression, and Characterization of a 13-Hydroperoxide Lyase Gene from Olive Fruit Related to the Biosynthesis of the Main Virgin Olive Oil Aroma Compounds

A full-length cDNA clone (OepHPL) coding for hydroperoxide lyase was isolated from olive fruit ( Olea europaea cv. Picual). The deduced amino acid sequence shows significant similarity to known plant hydroperoxide lyases and contains a N-terminal sequence that displays structural features of a chloroplast transit peptide. Genomic Southern blot analysis indicates that at least one copy of OepHPL is present in the olive genome. The recombinant hydroperoxide lyase was specific for 13-hydroperoxide derivatives of linolenic and linoleic acids but did not use 9-hydroperoxy isomers as substrates. Analyses of reaction products revealed that this enzyme produces primarily (Z)-hex-3-enal, which partially isomerizes to (E)-hex-2-enal, from 13-hydroperoxylinolenic acid and hexanal from 13-hydroperoxylinoleic acid. Expression levels were measured in different tissues of Picual and Arbequina varieties, including mesocarp and seed during development and ripening of olive fruits. The involvement of this olive hydroperoxide lyase gene in the biosynthesis of virgin olive oil aroma compounds is discussed.

Nucleotide Selectivity of Antibiotic Kinases

Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3')-IIIa, APH(2'')-Ib, and MPH(2')-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3')-IIIa exclusively uses ATP, MPH(2')-I exclusively uses GTP, and APH(2'')-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance.

Assessment of the Enzymatic Activity and Inhibition using HPFA with a Microreactor, Trypsin, Absorbed on Immobilized Artificial Membrane

The purpose of this study was to develop a new method to assess the activity and inhibition of the immobilized enzyme trypsin based on the frontal analysis of enzymatic reaction products. This novel technique was performed by on-line monitoring of the absorption at 253 nm of N-benzoyl-L-arginine (BA) from the hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE). A microreactor was constructed by immobilizing trypsin on an immobilized artificial membrane (IAM)-packed column. Trypsin was non-covalently and dynamically immobilized on-line in the hydrophobic interface of an IAM liquid chromatographic stationary phase. The trypsin-IAM stationary phase was bioactive. Due to the enzymatic reaction, the substrate of BAEE was completely hydrolyzed when the BAEE concentration was below 0.5 mmol/L and partly hydrolyzed when the BAEE concentration ranged from 0.75 to 1.0 mmol/L. By the addition of soybean trypsin inhibitor (STI), phenylmethane sulfonyl fluoride (PMSF), and benzamidine into the substrate solution, the results obtained from the frontal analysis showed that the activity of trypsin on IAM was strongly inhibited not only by STI but by both benzamidine and PMSF.

Modelling the bacterial communities associated with cystic fibrosis lung infections

In many human diseases that cystic fibrosis (CF) patients suffer from, for example, lung infections, bacteria have been considered to grow as biofilms. The ability of key CF pathogens such as Pseudomonas aeruginosa to resist antibiotic therapies may be due to the poor drug penetration of these biofilms. The overall aim of this study was to develop biofilm models in vitro that resembled the bacterial species composition of CF sputa. Here, this was a step towards a longer term goal of forming multiple bacterial biofilm models in vitro that would serve, in turn, as better assays of antibiotic susceptibilities than conventionally grown cells. Biofilm models were constructed from 31 CF sputum samples, using a modified microtitre plate assay. Three forms of assessment of these biofilms were made, namely, the mass, microscopic analysis and species composition. Species composition in sputa and biofilms, characterised by terminal restriction fragment length polymorphism (T-RFLP) analysis of ribosomal gene polymerase chain reaction (PCR) products amplified from directly extracted nucleic acids, indicated that the bacterial community in sputa was well reproduced in the biofilm models. Typically, fresh sputa contained 4.6 +/- 2.3 bacterial species, with the species number decreasing to 4.0 +/- 1.6 over 5 days-this was not statistically significant (p = 0.29). This study outlines a novel methodology by which to generate and study bacterial biofilms communities. It is also hoped that the versatility of this in vitro approach, combined with its simplicity and high reproducibility, will make it an effective system to study CF sputum biofilm development and, in the longer term, serve as a means of assessing antibiotic susceptibilities.

Ultrahigh vacuum/high-pressure flow reactor for surface x-ray diffraction and grazing incidence small angle x-ray scattering studies close to conditions for industrial catalysis

A versatile instrument for the in situ study of catalyst surfaces by surface x-ray diffraction and grazing incidence small angle x-ray scattering in a 13 ml flow reactor combined with reaction product analysis by mass spectrometry has been developed. The instrument bridges the so-called "pressure gap" and "materials gap" at the same time, within one experimental setup. It allows for the preparation and study of catalytically active single crystal surfaces and is also equipped with an evaporator for the deposition of thin, pure metal films, necessary for the formation of small metal particles on oxide supports. Reactions can be studied in flow mode and batch mode in a pressure range of 100-1200 mbar and temperatures up to 950 K. The setup provides a unique combination of sample preparation, characterization, and in situ experiments where the structure and reactivity of both single crystals and supported nanoparticles can be simultaneously determined.

A highly efficient growth mechanism of polycyclic aromatic hydrocarbons

A highly efficient growth mechanism of polycyclic aromatic hydrocarbons (PAHs) initiated and accelerated by phenyl radicals has been investigated on the basis of kinetic analysis of gas phase reaction products of pyrolysis of benzene with and without addition of acetylene and acetylene only. Pyrolytic reactions were performed in a flow tube reactor and the resulting products were detected by an in situ direct sampling mass spectrometric technique using a vacuum ultraviolet (VUV) single photon ionization (SPI) time of flight mass spectrometry (TOFMS). The detected species varies from smaller to larger PAHs up to m/z = 454 (C(36)H(22)) including primary PAHs, polyphenyl-PAHs and cyclopentafused-PAHs (CP-PAHs). The peculiarity of this result is an appearance of mass peaks at regular mass number intervals of approximately 76 that correspond to phenyl-PAHs produced by phenyl radical addition (+C(6)H(5), +77) followed by hydrogen elimination (-H, -1). All such mass peaks were found diminishing with appearance of -2 mass number peaks with increasing temperatures, certainly due to a conversion of thermally rather unstable phenyl-PAHs into stable condensed PAHs through a dehydrocyclization (-H(2), -2) process. In the same way, in the case of only acetylene pyrolysis, mass peaks at regular mass number intervals of 24 corresponding to the HACA (hydrogen abstraction/C(2)H(2) addition) products, were observed. Kinetic analysis of formation pathways of those observed products showed the active role of PAC (phenyl addition/cyclization) because of its efficiency to continue the endless growth of PAHs, while the HACA was only found efficient for producing symmetrical PAHs by filling a triple fusing site (four carbon bay structure). Especially, acetylene was mixed with benzene to understand the impact of HACA on the PAC path ways that resulted in enhancement of phenyl-PAHs production in spite of trapping of active and chain carrier species phenyl radicals by C(2)H(2) to form phenylacetylene. The comparison of HACA and PAC concluded that PAC is a highly efficient mechanism for the growth of PAHs and lastly their combined roles in combustion have been discussed. Hopefully, PAC will be useful to understand the process of aromatic growth, from furnaces to stellar atmospheres.

Biochemical and structural characterization of CYP124: a methyl-branched lipid omega-hydroxylase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) produces a variety of methyl-branched lipids that serve important functions, including modulating the immune response during pathogenesis and contributing to a robust cell wall that is impermeable to many chemical agents. Here, we report characterization of Mtb CYP124 (Rv2266) that includes demonstration of preferential oxidation of methyl-branched lipids. Spectrophotometric titrations and analysis of reaction products indicate that CYP124 tightly binds and hydroxylates these substrates at the chemically disfavored omega-position. We also report X-ray crystal structures of the ligand-free and phytanic acid-bound protein at a resolution of 1.5 A and 2.1 A, respectively, which provide structural insights into a cytochrome P450 with predominant omega-hydroxylase activity. The structures of ligand-free and substrate-bound CYP124 reveal several differences induced by substrate binding, including reorganization of the I helix and closure of the active site by elements of the F, G, and D helices that bind the substrate and exclude solvent from the hydrophobic active site cavity. The observed regiospecific catalytic activity suggests roles of CYP124 in the physiological oxidation of relevant Mtb methyl-branched lipids. The enzymatic specificity and structures reported here provide a scaffold for the design and testing of specific inhibitors of CYP124.

Analysis of products from the oxidation of technical lignins by oxygen and H3PMo12O40 in water and aqueous methanol by size-exclusion chromatography

Abstract One kraft lignin and two lignosulfonates were oxidized in aqueous acidic solutions containing a polyoxometalate (POM). The degradations were carried out in H2O or MeOH/H2O mixtures in the presence of oxygen. The treatment with aqueous H3PMo12O40 led to the dissolution of the studied lignins in the acidic medium (pH 1–2) and to the formation of up to 6.5 wt% vanillin and 6.2 wt% methyl vanillate based on the weight of dry lignin. The lignin oxidation products were analyzed by size-exclusion chromatography (SEC). For this purpose, a SEC method was developed, which allows the analysis of kraft lignin, lignosulfonates, and reaction products thereof without the need to remove the homogeneous catalyst. This method allows the direct observation of depolymerization and repolymerization reactions and hence the provision of a tool for studying the underlying chemistry. It has been demonstrated that the depolymerization of kraft lignin in water is accompanied by counterproductive condensation reactions. These repolymerization reactions were effectively prevented by addition of methanol, which couples competitively with lignin intermediates.

Comparing the Southern blot method and polymerase chain reaction product analysis for chimeric RCCX detection in CYP21A2 deficiency

The 3.2-kb TaqI-produced fragment of the CYP21A1P pseudogene and the 3.7-kb TaqI-produced fragment of the functional CYP21A2 gene exist on chromosome 6p21.3. We used the polymerase chain reaction (PCR) product and Southern blot method with TaqI endonuclease digestion to identify a chimeric RCCX module in two unrelated patients with congenital adrenal hyperplasia (CAH). After TaqI cleavage, the PCR product analysis revealed that patient 1 with the chimeric CYP21A1P/ CYP21A2 gene in one allele and IVS2-12A/C>G in combination with the 707-714del mutation in the other allele produced a configuration of 3.2- and 2.4-kb fragments. Patient 2, who carried IVS2-12A/C>G in combination with the 707-714del mutation in one allele and the chimeric TNXA/ TNXB gene in the other allele, presented with 3.2- and 2.3-kb fragments. However, Southern blot analysis showed that patients 1 and 2 produced 3.2-, 2.4-, and 2.5-kb fragments. We conclude that the chimeric CYP21A1P/ CYP21A2 gene, IVS2-12A/C>G in combination with the 707-714del mutation, and the chimeric TNXA/ TNXB gene cannot be distinguished by the Southern blot method. Conversely, the chimeric TNXA/ TNXB gene was identified in the PCR product analysis due to the appearance of the 2.37-kb fragment, which indicates the occurrence of the chimeric TNXA/ TNXB formation extending to the boundary of TNXA in the RCCX region.

Functional Characterization of Two 13-Lipoxygenase Genes from Olive Fruit in Relation to the Biosynthesis of Volatile Compounds of Virgin Olive Oil

Two LOX cDNA clones, Oep1LOX2 and Oep2LOX2, have been isolated from olive ( Olea europaea cv. Picual). Both deduced amino acid sequences showed significant similarity to known plant LOX2, and they contain an N-terminal chloroplastic transit peptide. Genomic Southern blot analyses suggest that at least three copies of Oep1LOX2 and one copy of Oep2LOX2 should be present in the olive genome. Linolenic acid proved to be the preferred substrate for both olive recombinant LOXs, and analyses of reaction products revealed that both enzymes produce primarily 13-hydroperoxides from linoleic and linolenic acids. Expression levels of both genes were measured in the mesocarp and seeds during development and ripening of Picual and Arbequina olive fruit along with the level of volatile compounds in the corresponding virgin olive oils. Biochemical and gene expression data suggest a major involvement of the Oep2LOX2 gene in the biosynthesis of virgin olive oil aroma compounds.

Multiple isoforms of PAX5 are expressed in both lymphomas and normal B‐cells

The transcription factor Pax5 plays a critical role in B cell development. It has been shown that alternative splicing of its gene (PAX5) produces several distinct transcripts that modify the amino acid sequence of the putative Pax5 proteins. Subsequent studies have attempted to correlate the expression of PAX5 isoforms with certain B-cell lymphomas, the conclusions of which suggest that altered isoform expression is involved in lymphomagenesis. However, in the absence of definitive data for PAX5 isoform expression patterns in normal B cells it is difficult to confirm whether aberrant isoform expression can indeed be correlated with disease. Using a high-resolution method of analysis of reverse transcription polymerase chain reaction products, we sought to analyse the expression of the different PAX5 isoforms in normal B-cells as well as a number of B-cell lymphoma and chronic lymphocytic leukaemia cases. It was found that multiple PAX5 isoforms were expressed in both normal and malignant B cells. Immunodetection and polysomal RNA analyses also confirmed that the different PAX5 mRNAs were translated into their corresponding proteins. No consistent deregulation of PAX5 isoform expression was observed in B-cell lymphomas, but rather, complex isoform expression patterns were found in normal B cell as well as B-cell lymphoma and CLL cases.

Kinetics and mechanism of the dehydration reaction of sarcosine to a bislactame through diacyclperoxide intermediate in strong acidic medium

AbstractThe influence of substitution on the amine functional group of glycine in the permanganic oxidation of such an α‐amino acid in moderately concentrated sulfuric acid medium has been investigated. Reaction products analysis has revealed that contrary to the usual α‐amino acid oxidation product, which is an aldehyde species, a valuable compound, namely 1,4‐dimethylpiperazine‐2,5‐dione, has been obtained as the main product via a cheap, simple, efficient, and novel method. Sarcosine has been chosen as a substituted derivative of glycine, and the kinetics and mechanism of its permanganic oxidation have been investigated using a spectrophotometric technique. Conclusive evidence has proven delayed autocatalytic activity for Mn(II) in this reaction, analogous to some α‐amino acids. It has been revealed that such activity can show up when a certain concentration ratio of Mn(II) to sarcosine is built up in the medium, which we call the “critical ratio.” The magnitude of the latter ratio depends on the sulfuric acid concentration. Considering the “delayed autocatalytic behavior” of Mn(II) ions, rate equations satisfying observations for both catalytic and noncatalytic routes have been presented. The reaction shows first‐order dependence on permanganate ions and sarcosine concentrations in both catalytic and noncatalytic pathways, and apparent first‐order dependence on Mn2+ ions in catalytic pathways. The correspondence of pseudo‐order rate constants of the catalytic and noncatalytic pathways to Arrhenius and Eyring laws has verified “critical ratio” as well as “delayed autocatalytic behavior” concepts. The activation parameters associated with both pathways have been computed and discussed. Mechanisms for both catalytic and noncatalytic routes involving radical intermediates as well as a product having a diketopiperazine skeleton have been reported for the first time. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 41: 689–703, 2009

Alteration of microRNA expression in vinyl carbamate-induced mouse lung tumors and modulation by the chemopreventive agent indole-3-carbinol

MicroRNAs (miRNAs) are small, non-protein-coding RNAs that can function as tumor suppressors or oncogenes. Deregulation of miRNA expression has been reported in lung cancer. However, modulation of miRNA expression by chemopreventive agents remains to be defined. In the present study, we examined if the chemopreventive agent indole-3-carbinol (I3C) reversed vinyl carbamate (VC)-induced deregulation of miRNA levels in lung tissues of female A/J mice. Lung tissues were obtained from a previous chemoprevention study, in which mice were treated with VC and given I3C in the diet for 15 weeks. Microarray studies revealed alterations in the expression of a number of miRNAs in lung tumors relative to that of normal lungs. miR-21, mir-31, miR-130a, miR-146b and miR-377 were consistently upregulated, whereas miR-1 and miR-143 were downregulated in lung tumors relative to normal lungs. In mice treated with VC and given I3C in the diet, levels of miR-21, mir-31, miR-130a, miR-146b and miR-377 were reduced relative to the level in mice treated with the carcinogen only. The results of the microarray study were confirmed by quantitative reverse transcription-polymerase chain reaction and gel analysis of polymerase chain reaction products. Further studies with miR-21 indicated that phosphatase and tensin homolog, programmed cell death 4 and rich protein with Kazal motifs are potential targets for the oncogenic effect of miR-21 and the chemopreventive activity of I3C. Taken together, we showed here that miRNAs are deregulated during VC-induced mouse lung tumorigenesis and their levels are modulated by I3C. Therefore, miRNAs and their target genes are promising biomarkers for the diagnosis of lung cancer and efficacy of chemopreventive/chemotherapeutic agents.

A new look at determining acid absorption of lead oxide used in the manufacturing of Pb-acid batteries

Acid absorption is a routine analytical method used in the manufacturing of lead oxide, that is, then further used to manufacture Pb-acid battery electrodes. This study had a new look at the definition of acid absorption being an indication of the acid reactivity of the oxide forming certain lead sulphate-related phases. Quantitative powder X-ray diffraction analysis of the acid reaction products showed there were significant differences in the phases formed that could be related to the amount of acid used in the acid absorption test. The study also showed that there were significant changes in the surface area of the oxides once they had reacted with the acid, where a traditionally slow reacting oxide such as β-PbO would show the greatest increase in material surface area once reacted with the acid. The accuracy of the method used by various laboratories was also studied by comparing the results obtained from two different methods and from three different laboratories. The results showed that there were significant differences between the reported values, and that one should with caution compare acid absorption numbers obtained from different laboratories.

“Reverse degradomics”, monitoring of proteolytic trimming by multi‐CE and confocal detection of fluorescent substrates and reaction products

A platform for profiling of multiple proteolytic activities acting on one specific substrate, based on the use of a 96-channel capillary DNA sequencer with CE-LIF of labeled substrate peptides and reaction products is introduced. The approach consists of synthesis of a substrate peptide of interest, fluorescent labeling of the substrate, either aminoterminally by chemical coupling, or carboxyterminally by transglutaminase reaction, proteolysis by a biological mixture of proteases in the absence or presence of protease inhibitors, multi-channel analysis of substrate and reaction products, and data collection and processing. Intact substrate and reaction products, even when varying by only one amino acid, can be relatively semi-quantified in a high-throughput manner, yielding information on proteases acting in complex biological mixtures and without prepurification. Monitoring, classification and inhibition of multiple proteolytic activities are demonstrated on a model substrate, the aminoterminus of the mouse granulocyte chemotactic protein-2. In view of extensive processing of chemokines into various natural forms with different specific biological activities, and of the fragmentary knowledge of processing proteases, examples of processing by neutrophil degranulate, tumor cell culture fluids and plasma are provided. An example of selection and comparison of inhibitory mAbs illustrates that the platform is suitable for inhibitor screening. Whereas classical degradomics technologies analyze the substrate repertoire of one specific protease, here the complementary concept, namely the study of all proteases acting, in a biological context, on one specific substrate, is developed and tuned to identify key proteases and protease inhibitors for the processing of any biological substrate of interest.

Human immunodeficiency virus–associated precursor T-lymphoblastic leukemia/lymphoblastic lymphoma: report of a case and review of the literature

We describe a case of human immunodeficiency virus-associated T-lymphoblastic leukemia/lymphoblastic lymphoma in a 43-year-old Italian man with a history of human immunodeficiency virus infection lasting 9 years. Immunoperoxidase stains showed that neoplastic cells were positive for CD3, TdT, CD45, CD10, CD1a, CD2, CD7, CD5, and CD43 (focal). The proliferation rate was approximately 70%, assessed by Ki-67/MIB-1 staining. Flow cytometry of the marrow aspirate revealed an intermediate/cortical T-lymphoblastic phenotype: negative for surface CD3 and positive for cytoplasmic CD3, CD1a, TdT, CD2, CD7, CD5, and CD8, with partial coexpression of dimCD4. Analysis of T-cell receptor gamma polymerase chain reaction products showed clonality. T-lymphoblastic leukemia/lymphoblastic lymphoma is a very rare occurrence in the clinical setting of human immunodeficiency virus infection. It is not listed in the World Health Organization classification of lymphomas associated with human immunodeficiency virus infection. Only 4 cases of human immunodeficiency virus-associated T-lymphoblastic leukemia/lymphoblastic lymphoma are reported in the current medical literature.

The structural basis for catalytic function of GMD and RMD, two closely related enzymes from the GDP‐d‐rhamnose biosynthesis pathway

The rare 6-deoxysugar D-rhamnose is a component of bacterial cell surface glycans, including the D-rhamnose homopolymer produced by Pseudomonas aeruginosa, called A-band O polysaccharide. GDP-D-rhamnose synthesis from GDP-D-mannose is catalyzed by two enzymes. The first is a GDP-D-mannose-4,6-dehydratase (GMD). The second enzyme, RMD, reduces the GMD product (GDP-6-deoxy-D-lyxo-hexos-4-ulose) to GDP-d-rhamnose. Genes encoding GMD and RMD are present in P. aeruginosa, and genetic evidence indicates they act in A-band O-polysaccharide biosynthesis. Details of their enzyme functions have not, however, been previously elucidated. We aimed to characterize these enzymes biochemically, and to determine the structure of RMD to better understand what determines substrate specificity and catalytic activity in these enzymes. We used capillary electrophoresis and NMR analysis of reaction products to precisely define P. aeruginosa GMD and RMD functions. P. aeruginosa GMD is bifunctional, and can catalyze both GDP-d-mannose 4,6-dehydration and the subsequent reduction reaction to produce GDP-D-rhamnose. RMD catalyzes the stereospecific reduction of GDP-6-deoxy-D-lyxo-hexos-4-ulose, as predicted. Reconstitution of GDP-D-rhamnose biosynthesis in vitro revealed that the P. aeruginosa pathway may be regulated by feedback inhibition in the cell. We determined the structure of RMD from Aneurinibacillus thermoaerophilus at 1.8 A resolution. The structure of A. thermoaerophilus RMD is remarkably similar to that of P. aeruginosa GMD, which explains why P. aeruginosa GMD is also able to catalyze the RMD reaction. Comparison of the active sites and amino acid sequences suggests that a conserved amino acid side chain (Arg185 in P. aeruginosa GMD) may be crucial for orienting substrate and cofactor in GMD enzymes.

Chlorella Starch Branching Enzyme II (BEII) Can Complement the Function of BEIIb in Rice Endosperm

In monocots, starch branching enzyme II (BEII) was functionally differentiated into BEIIa and BEIIb after separation from the dicots, and in cereals BEIIb plays a distinct role in amylopectin biosynthesis in the endosperm. The present study was conducted to examine to what extent a green algal BEII has an overlapping function with BEIIb in starch biosynthesis by introducing the Chlorella BEII gene into an amylose-extender (ae) mutant of rice. Chlorella BEII was found to complement the contribution of the rice endosperm BEIIb to the structures of amylopectin and starch granules because these mutated phenotypes were recovered almost completely to those of the wild type by the expression of Chlorella BEII. When the recombinant BE enzymes were incubated with the rice ae amylopectin, the branching pattern of Chlorella BEII was much more similar to that of rice BEIIb rather than rice BEIIa. Detailed analyses of BE reaction products suggests that BEIIb and Chlorella BEII only transfer chains with a degree of polymerization (DP) of 6 and 7, whereas BEIIa preferably transfers short chains with a DP of about 6-11. These results show that the Chlorella BEII is functionally similar to rice BEIIb rather than BEIIa.

O -Methyltransferase from Soybean Uses Both Caffeoyl-CoA and Flavonoids as Substrates

A gene encoding O-methyltransferase (SOMT)-10 from soybean, SOMT-10, was cloned by reverse transcription polymerase chain reaction. Phylogenetic analysis revealed that SOMT-10 belonged to caffeoyl-CoA O-methyltransferase (CCoAOMT) and contained conserved catalytic residues found in CCoAOMT. SOMT-10 was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified to determine its substrate. Several compounds including caffeoyl-CoA, naringenin, quercetin, caffeic acid, kaempferol and luteonin were tested as substrates for the purified recombinant SOMT-10. Analysis of reaction products using high performance liquid chromatography revealed that SOMT-10 used caffeoyl-CoA, quercetin and luteolin as substrates. This result indicated that SOMT-10 used flavones having vicinal hydroxyl groups. The methylation position was determined to be the 3′ hydroxyl group. It is likely that SOMT-10 is a new class of OMT that uses not only caffeoyl-CoA, but also flavonoids. Molecular docking of tricetin with the modeled structure SOMT-10 disclosed that SOMT-10 showed all combinations of the O-methylated products.