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Second Enzyme (second + enzyme)
Selected AbstractsAmperometric Biosensors Based on Choline Oxidase Entrapped in Polyacrylamide MicrogelsELECTROANALYSIS, Issue 2-3 2007López, M. Sánchez-Paniagua Abstract A choline amperometric biosensor has been designed using as biological component choline oxidase (ChOx) entrapped in polyacrylamide microgels. The working electrode was prepared by holding the enzyme loaded microgels on a platinum electrode by a dialysis membrane. It was found that the optimum microgel cross-linking required to retain ChOx and to allow the diffusion of choline was 7.0%. The response of the biosensor was optimized in relation to pH, temperature and working potential and the following optimal values were obtained: pH,9.0, temperature range between 20 and 30,°C, and potential +0.6,V. Under optimal conditions the sensitivity for choline was 17.45,mA M,1 cm,2, the detection limit 8,,M, and the response linear range from 2×10,5 M to 2×10,4 M. This biosensor has been also used as a nicotine detector due to the inhibition of the catalytic activity of choline oxidase by this compound. Moreover, the simultaneous entrapment of a second enzyme, acetylcholinesterase (AChE), in the microgels makes the biosensor sensible to acetylcholine. [source] Fitness drift of an atrazine-degrading population under atrazine selection pressureENVIRONMENTAL MICROBIOLOGY, Issue 3 2008Marion Devers Summary Pseudomonas sp. ADP harbouring the atrazine catabolic plasmid ADP1 was subcultured in liquid medium containing atrazine as sole source of nitrogen. After approximately 320 generations, a new population evolved which replaced the initial population. This newly evolved population grew faster and degraded atrazine more rapidly than the initial population. Plasmid profiles and Southern blot analyses revealed that the evolved strain, unlike the ancestral strain, presented a tandem duplication of the atzB gene encoding the second enzyme of the atrazine catabolic pathway responsible for the transformation of hydroxyatrazine to N-isopropylammelide. This duplication resulted from a homologous recombination that occurred between two direct repeats of 6.2 kb flanking the atzB gene and constituted by the insertion sequences IS1071, ISPps1 and a pdhL homologous sequence. This study highlights the IS-mediated plasticity of atrazine-degrading potential and demonstrates that insertion sequences not only help to disperse the atrazine-degrading gene but also improve the fitness of the atrazine-degrading population. [source] Isolation of the dxr gene of Zymomonas mobilis and characterization of the 1-deoxy- D -xylulose 5-phosphate reductoisomeraseFEMS MICROBIOLOGY LETTERS, Issue 1 2000Sigrid Grolle Abstract The gene encoding the second enzyme of the 2C -methyl- D -erythritol 4-phosphate (MEP) pathway for isopentenyl diphosphate biosynthesis, 1-deoxy- D -xylulose 5-phosphate (DXP) reductoisomerase, was cloned and sequenced from Zymomonas mobilis. The deduced amino acid sequence showed the highest identity (48.2%) to the DXP reductoisomerase of Escherichia coli. Biochemical characterization of the purified DXP reductoisomerase showed a strict dependence of the enzyme on NADPH and divalent cations (Mn2+, Co2+ or Mg2+). The enzyme is a dimer with a molecular mass of 39 kDa per subunit and has a specific activity of 19.5 U mg protein,1. Catalysis of the intramolecular rearrangement and reduction of DXP to MEP is competitively inhibited by the antibiotic fosmidomycin with a Ki of 0.6 ,M. [source] Ontogenetic effects of MAO-A inhibition on rat pineal n -acetylserotonin and melatonin during the first month of neonatal lifeHUMAN PSYCHOPHARMACOLOGY: CLINICAL AND EXPERIMENTAL, Issue 8 2000Gregory F Oxenkrug Abstract Inhibitors of monoamine oxidase A (MAO-A) but not MAO-B stimulate the activity of pineal serotonin N -acetyltransferase (AANAT) in the adult rat pineal leading to increased formation of N -acetyl serotonin (NAS) and melatonin (MEL). The pineal gland of the neonatal rat has AANAT activity, but the second enzyme in melatonin biosynthesis, HIOMT (hydroxyindole- O -methyltransferase) converting NAS to MEL, is absent during the first week of neonatal life. In this study we examined the effects of acute clorgyline treatment in vitro and in vivo, on pineal indoles over the first month of neonatal life. The results show that clorgyline stimulates NAS production by pineal both in vitro and in vivo from day five on with a marked increase between day 14 and day 21. In contrast, MEL is not increased until day 21, with a sharp rise thereafter. Copyright © 2000 John Wiley & Sons, Ltd. [source] Inhibition of human squalene monooxygenase by selenium compoundsJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2002Nisha Gupta Abstract Selenosis in animals is characterized by a variety of neurological abnormalities, but the chemical species of selenium and the molecular targets that mediate this neurotoxicity are unknown. We have previously shown that selenite is a potent inhibitor of squalene monooxygenase, the second enzyme in the committed pathway for cholesterol biosynthesis; inhibition of this enzyme by dimethyltellurium leads to a peripheral demyelinating neuropathy similar to that seen in selenosis. To evaluate the role methylation plays in selenium toxicity, we examined the ability of three methylselenium compounds, methylselenol, dimethylselenide, and trimethylselenonium iodide, to inhibit purified recombinant human squalene monooxygenase. IC50 values for methylselenol (95 ,M) and dimethylselenide (680 ,M) were greater than that previously obtained for selenite (37 ,M), and inhibition by trimethylselenonium iodide was evident only at concentrations above 3 mM. Inhibition by methylselenol as well as by selenite was slow and irreversible, suggestive of covalent binding to the enzyme, and thiol-containing compounds could prevent and reverse this inhibition, indicating that these compounds were reacting with sulfhydryl groups on the protein. Monothiols such as glutathione and ,-mercaptoethanol provided better protection than did dithiols, suggesting that these selenium compounds bind to only one of the two proposed vicinal cysteines on squalene monooxygenase. Unexpectedly, the inhibition by selenite was significantly enhanced by dithiols, indicating that a more toxic species, possibly selenide, was formed in the presence of these dithiol reductants. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:18,23, 2002; DOI 10.1002/jbt.10014 [source] Mutations in the cinnamate 4-hydroxylase gene impact metabolism, growth and development in ArabidopsisTHE PLANT JOURNAL, Issue 5 2009Anthony L. Schilmiller Summary The initial reactions of the phenylpropanoid pathway convert phenylalanine to p -coumaroyl CoA, a branch point metabolite from which many phenylpropanoids are made. Although the second enzyme of this pathway, cinnamic acid 4-hydroxylase (C4H), is well characterized, a mutant for the gene encoding this enzyme has not yet, to our knowledge, been identified, presumably because knock-out mutations in this gene would have severe phenotypes. This work describes the characterization of an allelic series of Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, each of which harbor mis-sense mutations in C4H (At2g30490). Heterologous expression of the mutant proteins in Escherichia coli yields enzymes that exhibit P420 spectra, indicative of mis-folded proteins, or have limited ability to bind substrate, indicating that the mutations we have identified affect protein stability and/or enzyme function. In agreement with the early position of C4H in phenylpropanoid metabolism, ref3 mutant plants accumulate decreased levels of several different classes of phenylpropanoid end-products, and exhibit reduced lignin deposition and altered lignin monomer content. Furthermore, these plants accumulate a novel hydroxycinnamic ester, cinnamoylmalate, which is not found in the wild type. The decreased C4H activity in ref3 also causes pleiotropic phenotypes, including dwarfism, male sterility and the development of swellings at branch junctions. Together, these observations indicate that C4H function is critical to the normal biochemistry and development of Arabidopsis. [source] The 1.25,Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosisACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2008Farah Javid-Majd Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79,Å. The structure of the apoenzyme reveals that the protein is composed of five ,-helices with connecting loops and is a member of the ,-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between ,-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily. [source] Crystallization and preliminary X-ray analysis of aspartate transcarbamoylase from the parasitic protist Trypanosoma cruziACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Kazuaki Matoba Aspartate transcarbamoylase (ATCase), the second enzyme of the de novo pyrimidine-biosynthetic pathway, catalyzes the production of carbamoyl aspartate from carbamoyl phosphate and l -aspartate. In contrast to Escherichia coli ATCase and eukaryotic CAD multifunctional fusion enzymes, Trypanosoma cruzi ATCase lacks regulatory subunits and is not part of the multifunctional fusion enzyme. Recombinant T. cruzi ATCase expressed in E. coli was purified and crystallized in a ligand-free form and in a complex with carbamoyl phosphate at 277,K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. Ligand-free crystals (space group P1, unit-cell parameters a = 78.42, b = 79.28, c = 92.02,Å, , = 69.56, , = 82.90, , = 63.25°) diffracted X-rays to 2.8,Å resolution, while those cocrystallized with carbamoyl phosphate (space group P21, unit-cell parameters a = 88.41, b = 158.38, c = 89.00,Å, , = 119.66°) diffracted to 1.6,Å resolution. The presence of two homotrimers in the asymmetric unit (38,kDa × 6) gives VM values of 2.3 and 2.5,Å3,Da,1 for the P1 and P21 crystal forms, respectively. [source] | |||||||||||||