1,720,967 research outputs found
Catalytic activity of the anaerobic tyrosine lyase required for thiamine biosynthesis in Escherichia coli
No full textThiazole synthase in Escherichia coli is an ?? heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the C?–C? bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cyclization reaction catalyzed by ThiG. ThiH is a radical S-adenosylmethionine (AdoMet) enzyme that utilizes a [4Fe-4S]+ cluster to reductively cleave AdoMet, forming methionine and a 5?-deoxyadenosyl radical. Analysis of the time-dependent formation of the reaction products 5?-deoxyadenosine (DOA) and p-cresol has demonstrated catalytic behavior of the tyrosine lyase. The kinetics of product formation showed a pre-steady state burst phase, and the involvement of DOA in product inhibition was identified by the addition of 5?-methylthioadenosine/S-adenosylhomocysteine nucleosidase to activity assays. This hydrolyzed the DOA and changed the rate-determining step but, in addition, substantially increased the uncoupled turnover of AdoMet. Addition of glyoxylate and ammonium inhibited the tyrosine cleavage reaction, but the reductive cleavage of AdoMet continued in an uncoupled manner. Tyrosine analogues were incubated with ThiGH, which showed a strong preference for phenolic substrates. 4-Hydroxyphenylpropionic acid analogues allowed uncoupled AdoMet cleavage but did not result in further reaction (C?–C? bond cleavage). The results of the substrate analogue studies and the product inhibition can be explained by a mechanistic hypothesis involving two reaction pathways, a product-forming pathway and a futile cycle
Controlled delivery of calcium gated MthK channels into an on-chip bilayer lipid membrane for electrophysiology measurements
No full textThe activity of a thermostable lipoyl synthase from Sulfolobus solfataricus with a synthetic octanoyl substrate
No full textThe protein lipoyl synthase (LipA) is essential for lipoic acid biosynthesis via sulfur insertions into a protein-bound octanoyl group. We have developed an in vitro assay for LipA using a synthetic tetrapeptide Substrate, containing an N-epsilon-octanoyl lysine residue, corresponding in sequence to the lipoyl binding domain of the E2 subunit of pyruvate dehydrogenase. A putative LipA from the hypothermophilic archaea Sulfolobus solfataricus was expressed in Escherichia coli and purified, and the activity was measured using this novel assay. The optimal temperature for the S. solfataricus LipA-dependent formation of the lipoyl group was found to be 60 degrees C
Bead-based immunoassays using a micro-chip flow cytometer
No full textA microfabricated flow cytometer has been developed for the analysis of micron-sized polymer beads onto which fluorescently labelled proteins have been immobilised. Fluorescence measurements were made on the beads as they flowed through the chip. Binding of antibodies to surface-immobilised antigens was quantitatively assayed using the device. Particles were focused through a detection zone in the centre of the flow channel using negative dielectrophoresis. Impedance measurements of the particles (at 703 kHz) were used to determine particle size and to trigger capture of the fluorescence signal. Antibody binding was measured by fluorescence at single and dual excitation wavelengths (532 nm and 633 nm). Fluorescence compensation techniques were implemented to correct for spectral overspill between optical detection channels. The data from the microfabricated flow cytometer was shown to be comparable to that of a commercial flow cytometer (BD-FACSAria). Graphical abstract image for this article (ID: b707507n
Multiplexed suspension array platform for high-throughput protein assays
No full textA multiplexed suspension array platform, based on SU8 disks patterned with machine-readable binary identification codes is presented. Multiple probe molecules, each attached to individual disks with different unique codes, provide multiplexed detection of targets in a small sample volume. The experimental system consists of a microfluidic chamber for arraying the particles in a manner suitable for high throughput imaging using a simple fluorescent microscope, together with custom software for automated code readout and analysis of assay response. The platform is demonstrated with a multiplexed antibody assay targeting 3 different human inflammatory cytokines. The suitability of the platform for other bio-analytical applications is discussed.<br/
Thiamine biosynthesis in Escherichia coli: Identification of the intermediate and by-product derived from tyrosine
No full textIn anaerobic organisms such as E. coli the tyrosine lyase ThiH is essential for the biosynthesis of the thiazole moiety of the vitamin thiamine. ThiH is a member of the radical AdoMet family. The products formed by cleavage of tyrosine in vitro have been identified and suggest a radical-mediated cleavage resulting in p-cresol and dehydroglycine which is hydrolyzed to glyoxylate
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Sensors for chemical detection based on top-down fabricated polycrystalline silicon nanowires
No full textSemiconducting Silicon (Si) nanowires (NWs) have been widely investigated for their potential to function as highly sensitive and selective sensors for both chemical and biological purposes. A key point of this sensing method is to be real-time and label-free. Several interesting sensing assays have been demonstrated such as sensing of ions, proteins, DNA and viruses[1-3]. The available approaches of silicon nanowire fabrication usually use some advanced lithographic techniques i.e., deep-UV, electron-beam or nanoimprint lithography to pattern silicon nanowires on SOI wafers. Recently, spacer nanowires patterned by a conventional anisotropic dry etch were used to form transistors. While this approach has the advantage of CMOS-compatibility, these techniques are extremely expensive and accessible only to large-scale integrated circuit manufacturers. While this approach delivers a cheap route for nanowire definition, nanowire volume control across the wafer remains challenging as the nanowire sidewall region generally receives unwanted etching
Thiazole synthase from Escherichia coli: an investigation of the substrates and purified proteins required for activity in vitro
No full textThiamine is biosynthesized by combining two heterocyclic precursors. In Escherichia coli and other anaerobes, one of the heterocycles, 4-methyl-5-(-hydroxyethyl) thiazole phosphate, is biosynthesized from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine. Genetic evidence has identified thiH, thiG, thiS, and thiF as essential for thiazole biosynthesis in E. coli. In this paper, we describe the measurement of the thiazole phosphate-forming reaction using purified protein components. The activity is shown to require four proteins isolated as heterodimers: ThiGH and ThiFS. Reconstitution of the [4Fe-4S] cluster in ThiH was essential for activity, as was the use of ThiS in the thiocarboxylate form. Spectroscopic studies with ThiGH strongly suggested that S-adenosylmethionine (AdoMet) bound to the [4Fe-4S] cluster, which became more susceptible to reduction to the +1 state. Assays of thiazole phosphate formation showed that, in addition to the proteins, Dxp, tyrosine, AdoMet, and a reductant were required. The analysis showed that no more than 1 mol eq of thiazole phosphate was formed per ThiGH. Furthermore, for each mole of thiazole-P formed, 1 eq of AdoMet and 1 eq of tyrosine were utilized, and 1 eq of 5'-deoxyadenosine was produced. These results demonstrate that ThiH is a member of the "radical-AdoMet" family and support a mechanistic hypothesis in which AdoMet is reductively cleaved to yield a highly reactive 5'-deoxyadenosyl radical. This radical is proposed to abstract the phenolic hydrogen atom from tyrosine, and the resultant substrate radical cleaves to yield dehydroglycine, which is required by ThiG for the thiazole cyclization reaction
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