Biochemical characterization of human and murine isoforms of UDP-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine 2-epimerase/<Emphasis Type="Italic">N</Emphasis>-acetylmannosamine kinase (GNE)

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme for the biosynthesis of sialic acids, terminal components of glycoconjugates associated with a variety of physiological and pathological processes. Different protein isoforms of human and mouse GNE, deriving from splice variants, were predicted recently: GNE1 represents the GNE protein described in several studies before, GNE2 and GNE3 are proteins with extended and deleted N-termini, respectively. hGNE2, recombinantly expressed in insect and mamalian cells, displayed selective reduction of UDP-GlcNAc 2-epimerase activity by the loss of its tetrameric state, which is essential for full enzyme activity. hGNE3, which had to be expressed in Escherichia coli, only possessed kinase activity, whereas mGNE1 and mGNE2 showed no significant differences. Our data therefore suggest a role of GNE1 in basic supply of cells with sialic acids, whereas GNE2 and GNE3 may have a function in fine-tuning of the sialic acid pathway.     

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine metabolism in <Emphasis Type="Italic">Bifidobacterium</Emphasis>

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism. S. Foley and E. Stolarczyk contributed equally to this work     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.     

Identification of metabolites produced from <Emphasis Type="Italic">N</Emphasis>-phenylpiperazine by <Emphasis Type="Italic">Mycobacterium</Emphasis> spp

Mycobacterium sp. 7E1B1W and seven other mycobacterial strains known to degrade hydrocarbons were investigated to determine their ability to metabolize the piperazine ring, a substructure found in many drugs. Cultures were grown at 30°C in tryptic soy broth and dosed with 3.1 mM N-phenylpiperazine hydrochloride; samples were removed at intervals and extracted with ethyl acetate. Two metabolites were purified from each of the extracts by high-performance liquid chromatography; they were identified by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy as N-(2-anilinoethyl)acetamide and N-acetyl-N′-phenylpiperazine. The results show that mycobacteria have the ability to acetylate piperazine rings and cleave carbon-nitrogen bonds.     

<Emphasis Type="Italic">N</Emphasis>-halamine biocidal coatings

Novel N-halamine siloxane and epoxide coatings are described. The coatings can be rendered biocidal by exposure to dilute bleach. Once the bound chlorine is lost from the coatings, it can be regenerated by further exposure to dilute bleach. Synthetic schemes and biocidal efficacy data are presented. The stabilities of the bound chlorine on the surfaces are also addressed. Substrates employed include sand, textiles, and paint. Potential uses for the technology are discussed.     

A comparison of the neuronal dysfunction caused by <Emphasis Type="Italic">Drosophila</Emphasis> tau and human tau in a <Emphasis Type="Italic">Drosophila</Emphasis> model of tauopathies

Hyperphosphorylation and aggregation of tau into tangles is a feature of disorders such as Alzheimer’s disease and other Tauopathies. To model these disorders in Drosophila melanogaster, human tau has been over-expressed and a variety of phenotypes have been observed including neurotoxicity, disrupted neuronal and synaptic function and locomotor impairments. Neuronal dysfunction has been seen prior to neuronal death and in the absence of tangle formation. The Drosophila tau protein shares a large degree of homology with human tau but differs in the crucial microtubule binding domains. Although like human tau Drosophila tau can induce neurotoxicity, little is known about its ability to disrupt neuronal function. In this study we demonstrate that like human tau, over-expression of Drosophila tau results in disrupted axonal transport, altered neuromuscular junction morphology and locomotor impairments. This indicates that like human tau, over-expression of Drosophila tau compromises neuronal function despite significant differences in microtubule binding regions.     

Production of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine Using Recombinant Chitinolytic Enzymes

The pharmaceutically important compound N-acetylglucosamine (NAG), is used in various therapeutic formulations, skin care products and dietary supplements. Currently, NAG is being produced by an environment-unfriendly chemical process using chitin, a polysaccharide present in abundance in the exoskeleton of crustaceans, as a substrate. In the present study, we report the potential of an eco-friendly biological process for the production of NAG using recombinant bacterial enzymes, chitinase (CHI) and chitobiase (CHB). The treatment of chitin with recombinant CHI alone produced 8% NAG and 72% chitobiose, a homodimer of NAG. However, supplementation of the reaction mixture with another recombinant enzyme, CHB, resulted in approximately six fold increase in NAG production. The product, NAG, was confirmed by HPLC, TLC and ESI-MS studies. Conditions are being optimized for increased production of NAG from chitin.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Methods for syntheses of <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="Italic">DL</Emphasis>-aspartic acid derivatives

Summary. A novel practical method for the synthesis of N-methyl-DL-aspartic acid 1 (NMA) and new syntheses for N-methyl-aspartic acid derivatives are described. NMA 1, the natural amino acid was synthesized by Michael addition of methylamine to dimethyl fumarate 5. Fumaric or maleic acid mono-ester and -amide were regioselectively transformed into beta-substituted aspartic acid derivatives. In the cases of maleamic 11a or fumaramic esters 11b, the α-amide derivative 13 was formed, but hydrolysis of the product provided N-methyl-DL-asparagine 9 via base catalyzed ring closure to DL-α-methylamino-succinimide 4, followed by selective ring opening. Efficient methods were developed for the preparation of NMA-α-amide 13 from unprotected NMA via sulphinamide anhydride 15 and aspartic anhydride 3 intermediate products. NMA diamide 16 was prepared from NMA dimethyl ester 6 and methylamino-succinimide 4 by ammonolysis. Temperature-dependent side reactions of methylamino-succinimide 4 led to diazocinone 18, resulted from self-condensation of methylamino-succinimide via nucleophyl ring opening and the subsequent ring-transformation.     

Variation of acharan sulfate and monosaccharide composition and analysis of neutral <Emphasis Type="Italic">N</Emphasis>-glycans in African giant snail (<Emphasis Type="Italic">Achatina fulica</Emphasis>)

Acharan sulfate content from African giant snail (Achatina fulica) was compared in eggs and snails of different ages. Acharan sulfate was not found in egg. Acharan sulfate disaccharide →4)-α-d-GlcNpAc (1→4)-α-l-IdoAp2S(1→, analyzed by SAX (strong-anion exchange)–HPLC was observed soon after hatching and increases as the snails grow. Monosaccharide compositional analysis showed that mole % of glucosamine, a major monosaccharide of acharan sulfate, increased with age while mole % of galactose decreased with age. These results suggest that galactans represent a major energy source during development, while acharan sulfate appearing immediately after hatching, is essential for the snail growth. The structures of neutral N-glycans released from eggs by peptide N-glycosidase F (PNGase F), were next elucidated using ESI-MS/MS, MALDI-MS/MS, enzyme digestion, and monosaccharide composition analysis. Three types of neutral N-glycan structures were observed, truncated (Hex_2–4-HexNAc₂), high mannose (Hex_5–9-HexNAc₂), and complex (Hex₃-HexNAc_2–10) types. None showed core fucosylation.     

Purification and characterization of a <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase produced by <Emphasis Type="Italic">Talaromyces emersonii</Emphasis> during growth on algal fucoidan

A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (K_m value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH 5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg²⁺, Co²⁺ and Fe³⁺.     

Mutational analysis of the binding affinity and transport activity for<Emphasis Type="Italic"> N</Emphasis> -acetylglucosamine of the novel ABC transporter Ngc in the chitin-degrader<Emphasis Type="Italic"> Streptomyces olivaceoviridis</Emphasis>

The highly differentiated bacterium Streptomyces olivaceoviridis efficiently hydrolyses chitin, a highly abundant natural polysaccharide, to low molecular weight products including N-acetylglucosamine (NAG) and N,N -diacetylchitobiose (chitobiose). NAG is taken up by a PTS (phosphoenolpyruvate-dependent phosphotransferase system) which includes the PtsC2 protein, and via the ABC (ATP-binding cassette) transporter Ngc, which itself includes the substrate-binding protein NgcE. This is at present the only ABC transporter which is known to mediate specific uptake of NAG (K_m 0.48 M, V_max 1.3 nmol/min/mg dry weight) and is competitively inhibited by chitobiose (K_i 0.68 M). The latter finding suggests that the Ngc system transports both NAG and chitobiose efficiently. To identify amino acid residues required for the function of NgcE, either the wild-type or one of several mutant forms of the ngcE gene was introduced into the strain S. olivaceoviridis NgcE/PtsC1/PtsC2, which lacks both functional transport systems for NAG, and chromosomal recombinants were selected. Based on the in vivo transport parameters of the recombinants, and the in vitro binding characteristics of the corresponding purified proteins, the following conclusions can be drawn. (1) Replacement of the C-terminally located residue Y396 by A (Y396A) has little effect on ligand-binding or transport parameters. The W395A mutation also induced little change in the substrate affinity in vitro, but it led in vivo to a marked increase (11 fold) in K_m, and enhanced V_max (by 1.5 fold). (2) The amino acids Y201 and W280 both contribute (51% and 38%) to the ligand-binding capacity of NgcE. They are both very important for the in vivo function of the complete transport apparatus; strains expressing either Y201A or W280A show drastically (100 or 150 times) enhanced K_m values. (3) The concomitant presence of either Y200 and W280 or Y201 and W280 is essential for the function of NgcE. (4) Y201 is located within a tyrosyl-rich motif. This has been found to share some features with the ligand-binding site of amelogenins (enamel matrix proteins), which interact with NAG residues in glycoconjugates. In addition, it is distantly related to the ligand-binding site(s) in the plant-lectins UDA ( Urtica dioica agglutinin, specific for NAG and its oligomers) and WGA (wheat germ agglutinin, which recognises a motif comprising three consecutive NAG residues).Communicated by A. Kondorosi     

Metabolism of <Emphasis Type="Italic">N</Emphasis>-Methyl, <Emphasis Type="Italic">N</Emphasis>-propargylphenylethylamine: Studies with Human Liver Microsomes and cDNA Expressed Cytochrome P450 (CYP) Enzymes

Summary 1. We used an in vitro screening procedure and studies with individual human liver microsomes and cDNA-expressed CYP enzymes to investigate the metabolism of the putative neuroprotective drug N-methyl,N-propargyl-2-phenylethylamine (MPPE) to N-methylphenylethylamine (N-methylPEA) and N-propargylphenylethylamine (N-propargylPEA). 2. An electron-capture gas chromatographic procedure previously developed in our laboratories was used to measure the quantities of N-methylPEA and N-propargylPEA formed in the experiments with a single donor human liver microsome panel and cDNA expressed single CYP enzyme systems. The data were fitted to nonlinear regressions using Prism to determine kinetic constants. The results from a fluorogenic screen determined which cDNA-expressed single CYP enzymes were investigated. 3. CYP2B6, CYP2C19, and CYP2D6 all contributed to the formation of N-methylPEA, while only CYP2B6 catalyzed the formation of N-propargylPEA. The K _M and V _max values for N-propargylPEA formation were 290 ± 70 μM and 139±16 ng/mL/min. The values for formation of N-methylPEA were not determined from these experiments due to the complexity of fitting the data to a three-variable equation, but data on the time course of N-methylPEA formation are presented. 4. Catabolism of MPPE to N-methylPEA and N-propargylPEA is catalyzed by CYP enzymes. CYP2B6, 2C19 and 2D6 all contribute to the depropargylation of the parent compound, but only CYP2B6 also catalyzes demethylation. CYP2C19 was found to be the most active with respect to generation of N-methylPEA.     

Enhanced production of tropane alkaloids in <Emphasis Type="Italic">Scopolia parviflora</Emphasis> by introducing the <Emphasis Type="Italic">PMT</Emphasis> (putrescine <Emphasis Type="Italic">N</Emphasis>-methyltransferase) gene

Summary In wild-type Scopolia parvilfora (Solanaceae) tissues, only the roots express the enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53), which is the first specific precursor of the tropane alkaloids. Moreover, the tropanane alkaloid levels were the highest in the root (0.9 mg g⁻¹ on a dry weight basis), followed by the stem and then the leaves. We metabolically engineered S. parviflora by introducing the tobacco pmt gene into its genome by a binary vector system that employs disarmed Agrobacterium rhizogenes. The kanamycin-resistant hairy root lines were shown to bear the pmt gene and to overexpress its mRNA and protein product by at least two-fold, as determined by polymerase chain reaction (PCR) and Northern and Western blottings, respectively. The transgenic lines also showed higher PMT activity and were morphologically aberrant in terms of slower growth and the production of lateral roots. The overexpression of pmt markedly elevated the scopolamine and hyoscyamine levels in the transgenic lines that showed the highest pmt mRNA and PMT protein levels. Thus, overexpression of the upstream regulator of the tropane alkaloid pathway enhanced the biosynthesis of the final product. These observations may be useful in establishing root culture systems that generate large yields of tropane alkaloids. These authors contributed equally to this paper (co-first authors).     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Cloning,characterization, and transformation of the phosphoethanolamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase gene (<Emphasis Type="Italic">ZmPEAMT1</Emphasis>) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-l-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering. Note: Nucleotide sequence data are available in GenBank under the following accession numbers: maize (Zea mays, ZmPEAMT1, AY626156; ZmPEAMT2, AY103779); rice (Oryza sativa, OsPEAMT1/Os01g50030, NM_192178; OsPEAMT2/Os05g47540, XM_475841); wheat (Triticum aestivum, TaPEAMT, AY065971); Arabidopsis (Arabidopsis thaliana, AtNMT1/At3g18000, AY091683; AtNMT2/At1g48600, NM_202264; AtNMT3/At1g73600, NM_106018); oilseed rape (Brassica napus, BnPEAMT, AY319479), tomato (Lycopersicon esculentum, AF328858), spinach (Spinacia oleracea, AF237633).     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan