No abstract

A number of strictly conserved residues present in all three domains indicate that LASPO, SDH and FRD share the same overall folding topology. Many of these conserved residues are in the FAD-binding site and active centre, suggesting a similar catalytic mechanism. Thus, LASPO, SDH and FRD form a class of functionally and structurally related oxidoreductases that are all able to reduce fumarate and to oxidise a dicarboxylate substrate.

L-Aspartate oxidase is a monomeric flavoprotein that catalyzes the first step in the de novo biosynthetic pathway for pyridine nucleotide formation under both aerobic and anaerobic conditions. In spite of the physiological importance of this biosynthesis in particular in facultative aerobic organisms, such as Escherichia coli, little is known about the electron acceptor of reduced L-aspartate oxidase in the absence of oxygen. In this report, evidence is presented which suggests that in vitro fumarate can play such a role. L-Aspartate oxidase binds succinate and fumarate with K,, values of 0.24 mM and 0.22 mM, respectively. A competitive behaviour was observed for these two dicarboxylic acids towards iminoaspartate and sulfite ions. Photoreduction experiments suggest that fumarate and succinate bind at or close to the active site of the molecule. A new fumarate reductase activity of L-aspartate oxidase is reported using benzylviologen or L-aspartate as reductants and fumarate as oxidant. Steady-state kinetics for the oxidase and the fumarate reductase activity of L-aspartate oxidase were obtained using either fumarate or oxygen as electron acceptor and L-aspartate as electron donor. Finally, succinate was identified as the product of the L-aspartate :fumarate oxidoreductase activity using radiolabeled fumarate under anaerobic conditions. The results suggest that fumarate can be a valuable alternative to oxygen as a substrate for L-aspartate oxidase.Keywords: flavoprotein; L-aspartate oxidase ; FAD ; fumarate reductase ; photoreduction.L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol non-covalently bound FAD/mol protein. The molecule catalyzes the oxidation of L-aspartate to the corresponding i mnoacid and the reduction of oxygen to H,O, [I]. In E. coli, the enzyme is specified by the nadB gene and is one of the two components of the quinolinate synthase complex. The complex catalyzes the biosynthesis of quinolinate from L-aspartate and dihydroxyacetone phosphate in the de now biosynthetic pathway for pyridine nucleotide formation [ 2 ] . Quinolinate is subsequently converted to NAD via a metabolic sequence common to all organisms. The de n o w NAD biosynthesis has been termed an anaerobic pathway based upon the observation that a mutation in the gene coding for L-aspartate oxidase is expressed under both aerobic and anaerobic growth conditions [3]. In particular, although in vitro studies demonstrate that oxygen is the obligate electron acceptor for L-aspartate oxidase [I], the possibility was not excluded that in vivo, under anaerobic conditions, Laspartate oxidase utilizes an electron acceptor other than oxygen. However, as far as we know, this compound is still undefined.In facultative aerobic organisms like E. coli, fumaric acid can be utilized as the terminal electron acceptor for biological oxidation of various organic compounds. The succinate-fumarate couple plays a role as either oxidant or reductant for the respiratory chain. The two reactions are catalyzed by succinate dehydrogenase and fumar...

A new serine proteinase inhibitor, rapeseed trypsin inhibitor (RTI), has been isolated from rapeseed (Brassica napus var. oleifera) seed. The protein inhibits the catalytic activity of bovine /?-trypsin and bovine a-chymotrypsin with apparent dissociation constants of 3.0 x lo-"' M and 4.1 x lo-' M, at pH 8.0 and 21"C, respectively. The stoichiometry of both proteinase-inhibitor complexes is 1: 1. The amino acid sequence of RTI consists of 60 amino acid residues, corresponding to an M, of about 6.7 kDa. The PI-P,' reactive site bond has been tentatively identified at position A$-Ile". RTI shows no similarity to other serine proteinase inhibitors except the low molecular weight mustard trypsin inhibitor (MTI-2). RTI and MTI-2 could be members of a new class of plant serine proteinase inhibitors.

Recent advances in the field of cellular reprogramming have opened a route to studying the fundamental mechanisms underlying common neurological disorders. High‐density microelectrode‐arrays (HD‐MEAs) provide unprecedented means to study neuronal physiology at different scales, ranging from network through single‐neuron to subcellular features. In this work, HD‐MEAs are used in vitro to characterize and compare human induced‐pluripotent‐stem‐cell‐derived dopaminergic and motor neurons, including isogenic neuronal lines modeling Parkinson's disease and amyotrophic lateral sclerosis. Reproducible electrophysiological network, single‐cell and subcellular metrics are used for phenotype characterization and drug testing. Metrics, such as burst shape and axonal velocity, enable the distinction of healthy and diseased neurons. The HD‐MEA metrics can also be used to detect the effects of dosing the drug retigabine to human motor neurons. Finally, it is shown that the ability to detect drug effects and the observed culture‐to‐culture variability critically depend on the number of available recording electrodes.