Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe–4S] protein

AbstractThe last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen-sensitive [4Fe–4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical

A combined computational and experimental investigation of the [2Fe–2S] cluster in biotin synthase

Biotin synthase was the first example of what is now regarded as a distinctive enzyme class within the radical S-adenosylmethionine superfamily, the members of which use Fe/S clusters as the sulphur source in radical sulphur insertion reactions. The crystal structure showed that this enzyme contains a [2Fe–2S] cluster with a highly unusual arginine ligand, besides three normal cysteine ligands. However, the crystal structure is at such a low resolution that neither the exact coordination mode nor the role of this exceptional ligand has been elucidated yet, although it has been shown that it is not essential for enzyme activity. We have used quantum refinement of the crystal structure and combined quantum mechanical and molecular mechanical calculations to explore possible coordination modes and their influences on cluster properties. The investigations show that the protonation state of the arginine ligand has little influence on cluster geometry, so even a positively charged guanidinium moiety would be in close proximity to the iron atom. Nevertheless, the crystallised enzyme most probably contains a deprotonated (neutral) arginine coordinating via the NH group. Furthermore, the Fe···Fe distance seems to be independent of the coordination mode and is in perfect agreement with distances in other structurally characterised [2Fe–2S] clusters. The exceptionally large Fe···Fe distance found in the crystal structure could not be reproduced

Green Synthesis as a Simple and Rapid Route to Protein Modified Magnetic Nanoparticles for Use in the Development of a Fluorometric Molecularly Imprinted Polymer-Based Assay for Detection of Myoglobin.

We have developed a low-cost molecularly imprinted polymer (MIP)-based fluorometric assay to directly quantify myoglobin in a biological sample. The assay uses a previously unreported method for the development of microwave-assisted rapid synthesis of aldehyde functionalized magnetic nanoparticles, in just 20 minutes. The aldehyde functionalized nanoparticles have an average size of 7.5 nm ± 1.8 and saturation magnetizations of 31.8 emu g-1 with near-closed magnetization loops, confirming their superparamagnetic properties. We have subsequently shown that protein tethering was possible to the aldehyde particles, with 0.25 ± 0.013 mg of myoglobin adsorbed to 20 mg of the nanomaterial. Myoglobin-specific fluorescently tagged MIP (F-MIP) particles were synthesized and used within the assay to capture myoglobin from a test sample. Excess F-MIP was removed from the sample using protein functionalized magnetic nanoparticles (Mb-SPION), with the remaining sample analysed using fluorescence spectroscopy. The obtained calibration plot of myoglobin showed a linear correlation ranging from 60 pg mL-1 to 6 mg mL-1 with the limit of detection of 60 pg mL-1. This method was successfully used to detect myoglobin in spiked fetal calf serum, with a recovery rate of more than 93%

Cell and Tissue Imaging with Molecularly Imprinted Polymers

International audienc

Molecularly imprinted polymer nanomaterials and nanocomposites by controlled/living radical polymerization.

International audienceSince the pioneering work of Wulff and Mosbach more than 30 years ago, molecular imprinting of synthetic polymers has emerged as a robust and convenient way for synthesizing polymeric receptor materials bearing specific recognition sites for target molecules. The resulting materials, molecularly imprinted polymers (MIPs), are therefore commonly referred to as ‘plastic antibodies’. They are obtained by polymerizing a scaffold around a target, or a derivate thereof, which acts as a molecular template. MIPs have been successfully applied in many areas including affinity separation, immunoassays, chemical sensing, solid-phase extraction, drug delivery, cell and tissue imaging, direct synthesis and catalysis. In terms of affinity and selectivity, MIPs are on a par with biological receptors like antibodies, and this is accompanied by a superior chemical and physical stability, compatibility with organic media, reusability, easy engineering and low cost. These advantages represent the main reasons for the wide interest raised around molecularly imprinted materials. Mainly produced by free radical polymerization (FRP) of vinyl monomers, MIPs have also taken advantage of the introduction of controlled/living radical polymerization (CRP) techniques, which have literally transformed polymer chemistry over the last decade. This review describes the advantages arising from the use of CRP in synthesizing MIPs, both in terms of sheer binding properties as well as for their remarkabl