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Polcari D.,McGill University | Kwan A.,Montreal Neurological Institute | Van Horn M.R.,Montreal Neurological Institute | Danis L.,McGill University | And 4 more authors.
Analytical Chemistry | Year: 2014

At the synapse, d-serine is an endogenous co-agonist for the N-methyl-d-aspartate receptor (NMDAR). It plays an important role in synaptic transmission and plasticity and has also been linked to several pathological diseases such as schizophrenia and Huntington's. The quantification of local changes in d-serine concentration is essential to further understanding these processes. We report herein the development of a disk-shaped amperometric enzymatic biosensor for detection of d-serine based on a 25 μm diameter platinum disk microelectrode with an electrodeposited poly-m-phenylenediamine (PPD) layer and an R. gracilis d-amino acid oxidase (RgDAAO) layer. The disk-shaped d-serine biosensor is 1-5 orders of magnitude smaller than previously reported probes and exhibits a sensitivity of 276 μA cm -2 mM-1 with an in vitro detection limit of 0.6 μM. We demonstrate its usefulness for in vivo applications by measuring the release of endogenous d-serine in the brain of Xenopus laevis tadpoles. © 2014 American Chemical Society.


Molla G.,University deiiInsubria | Molla G.,Centro Interuniversitario Of Biotecnologie Proteiche | Nardini M.,University of Milan | Motta P.,University deiiInsubria | And 6 more authors.
Biochemical Journal | Year: 2014

The aaoSogene from Streptococcus oligofermentans encodes a 43 kDa flavoprotein, aminoacetone oxidase (SoAAO), which was reported to possess a low catalytic activity against several different L-amino acids; accordingly, it was classified as an Lamino acid oxidase. Subsequently, SoAAO was demonstrated to oxidize aminoacetone (a pro-oxidant metabolite), with an activity ∼25-fold higher than the activity displayed on L-lysine, thus lending support to the assumption of aminoacetone as the preferred substrate. In the present study, we have characterized the SoAAO structure-function relationship. SoAAO is an FAD-containing enzyme that does not possess the classical properties of the oxidase/dehydrogenase class of flavoproteins (i.e. no flavin semiquinone formation is observed during anaerobic photoreduction as well as no reaction with sulfite) and does not show a true L-amino acid oxidase activity. From a structural point of view, SoAAO belongs to a novel protein family composed of three domains: an α/β domain corresponding to the FAD-binding domain, a β-domain partially modulating accessibility to the coenzyme, and an additional α-domain. Analysis of the reaction products of SoAAO on aminoacetone showed 2,5-dimethylpyrazine as the main product; we propose that condensation of two aminoacetone molecules yields 3,6-dimethyl-2,5-dihydropyrazine that is subsequently oxidized to 2,5-dimethylpyrazine. The ability of SoAAO to bind two molecules of the substrate analogue O-methylglycine ligand is thought to facilitate the condensation reaction. A specialized role for SoAAO in the microbial defence mechanism related to aminoacetone catabolism through a pathway yielding dimethylpyrazine derivatives instead of methylglyoxal can be proposed. ©2014 Biochemical Society.


Chirivi C.,CNR Institute of Chemistry of Molecular Recognition | Fontana G.,Indena S.p.A. | Monti D.,CNR Institute of Chemistry of Molecular Recognition | Monti D.,Centro Interuniversitario Of Biotecnologie Proteiche | And 5 more authors.
Chemistry - A European Journal | Year: 2012

Laccase-catalysed oxidation of ergot alkaloids in the absence of chemical mediators allowed the unexpected isolation of the mono-hydroxylated derivatives of compounds 2-7. Structure determination by NMR techniques clearly indicated that hydroxylation took place at the C-4 benzylic position. Quite notably, the proposed protocol allowed, for the first time, functionalisation at the C-4 position of the ergoline skeleton. Depending on the absence or on the presence of a C-10 α-methoxy substituent, hydroxylation was either stereoselective (furnishing C-4α OH derivatives) or gave rise to a C-4α/C-4β OH mixture in a 2:1 ratio, respectively. Mild laccase-catalysed oxidation of ergot alkaloids allowed, for the first time, the functionalisation of the C-4 position of these molecules. Accordingly, C-4 hydroxy derivatives were isolated and characterised (see scheme). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Conti G.,University of Insubria | Pollegioni L.,University of Insubria | Pollegioni L.,Centro Interuniversitario Of Biotecnologie Proteiche | Molla G.,University of Insubria | And 3 more authors.
FEBS Journal | Year: 2014

Semi-synthetic cephalosporins are synthesized from the 7-amino cephalosporanic acid (7-ACA) nucleus produced from the antibiotic cephalosporin C (CephC). In recent years, a single-step enzymatic process in which CephC is directly converted into 7-ACA by a cephalosporin C acylase (CA) has attracted industrial interest because of the prospects of simplifying the process and reducing costs. CAs are members of the glutaryl acylase family that specifically use CephC as their substrate; however, known natural glutaryl acylases show very low activity on the antibiotic. We previously enhanced the catalytic efficiency on CephC of a glutaryl acylase from Pseudomonas N176 (named VAC) by a protein engineering approach, and solved the structures of the VAC, thus providing insight into the substrate binding and catalytic activity of CAs. However, the properties of such enzymes are not sufficient to encourage 7-ACA manufacturers to shift to single-step enzymatic conversion of CephC. Here, we combine structural knowledge, semi-rational design, computational approaches and evolution analysis to isolate VAC variants with altered substrate specificity (i.e. with a > 11 000-fold increase in specificity constant for CephC versus glutaryl-7-amino cephalosporanic acid, compared to wild-type) and with the highest kinetic efficiency so far obtained for a CA. Indeed, the H57βS-H70βS-L154βY VAC variant shows the highest conversion of CephC into 7-ACA under conditions resembling those used at industrial level because of its high kinetic efficiency and the absence of substrate or product inhibition effects, and may be suitable for industrial application of the mono-step process for CephC conversion. The single-step enzymatic process in which CephC is directly converted into 7-ACA by a CephC acylase attracted the industrial interest because of the prospects of simplifying the process and reducing costs. We isolated engineered VAC variants with altered substrate specificity and with the highest kinetic efficiency so far obtained for a CephC acylase: they appear suitable for the industrial application. © 2014 FEBS.

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