Ángeles Canales

Deciphering the genetic determinants for aerobic nicotinic acid degradation: The nic cluster from Pseudomonas putida KT2440

The aerobic catabolism of nicotinic acid (NA) is considered a model system for degradation of N-heterocyclic aromatic compounds, some of which are major environmental pollutants; however, the complete set of genes as well as the structural–functional relationships of most of the enzymes involved in this process are still unknown. We have characterized a gene cluster (nic genes) from Pseudomonas putida KT2440 responsible for the aerobic NA degradation in this bacterium and when expressed in heterologous hosts. The biochemistry of the NA degradation through the formation of 2,5-dihydroxypyridine and maleamic acid has been revisited, and some gene products become the prototype of new types of enzymes with unprecedented molecular architectures. Thus, the initial hydroxylation of NA is catalyzed by a two-component hydroxylase (NicAB) that constitutes the first member of the xanthine dehydrogenase family whose electron transport chain to molecular oxygen includes a cytochrome c domain. The Fe2+-dependent dioxygenase (NicX) converts 2,5-dihydroxypyridine into N-formylmaleamic acid, and it becomes the founding member of a new family of extradiol ring-cleavage dioxygenases. Further conversion of N-formylmaleamic acid to formic and maleamic acid is catalyzed by the NicD protein, the only deformylase described so far whose catalytic triad is similar to that of some members of the α/β-hydrolase fold superfamily. This work allows exploration of the existence of orthologous gene clusters in saprophytic bacteria and some pathogens, where they might stimulate studies on their role in virulence, and it provides a framework to develop new biotechnological processes for detoxification/biotransformation of N-heterocyclic aromatic compounds.

The Bound Conformation of Microtubule-Stabilizing Agents : NMR Insights into the Bioactive 3D Structure of Discodermolide and Dictyostatin

A protocol based on a combination of NMR experimental data with molecular mechanics calculations and docking procedures has been employed to determine the microtubule-bound conformation of two microtubule-stabilizing agents, discodermolide (DDM) and dictyostatin (DCT). The data indicate that tubulin in assembled microtubules recognizes DDM through a conformational selection process, with minor changes in the molecular skeleton between the major conformer in water solution and that bound to assembled microtubules. For DCT, the deduced bound geometry presents some key conformation differences around certain torsion angles, with respect to the major conformer in solution, and still displays mobility even when bound. The bound conformer of DCT resembles that of DDM and provides very similar contacts with the receptor. Competition experiments indicate that both molecules compete with the taxane-binding site. A model of the binding mode of DDM and DCT to tubulin is proposed.

Solution NMR structure of a human FGF-1 monomer, activated by a hexasaccharide heparin-analogue

The 3D structure of a complex formed by the acidic fibroblast growth factor (FGF‐1) and a specifically designed synthetic heparin hexasaccharide has been determined by NMR spectroscopy. This hexasaccharide can substitute natural heparins in FGF‐1 mitogenesis assays, in spite of not inducing any apparent dimerization of the growth factor. The use of this well defined synthetic heparin analogue has allowed us to perform a detailed NMR structural analysis of the heparin–FGF interaction, overcoming the limitations of NMR to deal with the high molecular mass and heterogeneity of the FGF‐1 oligomers formed in the presence of natural heparin fragments. Our results confirm that glycosaminoglycans induced FGF‐1 dimerization either in a cis or trans disposition with respect to the heparin chain is not an absolute requirement for biological activity.

Zampanolide, a Potent New Microtubule-Stabilizing Agent, Covalently Reacts with the Taxane Luminal Site in Tubulin α,β-Heterodimers and Microtubules

Zampanolide and its less active analog dactylolide compete with paclitaxel for binding to microtubules and represent a new class of microtubule-stabilizing agent (MSA). Mass spectrometry demonstrated that the mechanism of action of both compounds involved covalent binding to β-tubulin at residues N228 and H229 in the taxane site of the microtubule. Alkylation of N228 and H229 was also detected in α,β-tubulin dimers. However, unlike cyclostreptin, the other known MSA that alkylates β-tubulin, zampanolide was a strong MSA. Modeling the structure of the adducts, using the NMR-derived dactylolide conformation, indicated that the stabilizing activity of zampanolide is likely due to interactions with the M-loop. Our results strongly support the existence of the luminal taxane site of microtubules in tubulin dimers and suggest that microtubule nucleation induction by MSAs may proceed through an allosteric mechanism.

A Reversible and Selective Inhibitor of Monoacylglycerol Lipase Ameliorates Multiple Sclerosis

Monoacylglycerol lipase (MAGL) is the enzyme responsible for the inactivation of the endocannabinoid 2-arachidonoylglycerol (2-AG). MAGL inhibitors show analgesic and tissue-protecting effects in several disease models. However, the few efficient and selective MAGL inhibitors described to date block the enzyme irreversibly, and this can lead to pharmacological tolerance. Hence, additional classes of MAGL inhibitors are needed to validate this enzyme as a therapeutic target. Here we report a potent, selective, and reversible MAGL inhibitor (IC50=0.18 μM) which is active in vivo and ameliorates the clinical progression of a multiple sclerosis (MS) mouse model without inducing undesirable CB1 -mediated side effects. These results support the interest in MAGL as a target for the treatment of MS.

Unravelling the gallic acid degradation pathway in bacteria: the gal cluster from Pseudomonas putida

Gallic acid (3,4,5‐trihydroxybenzoic acid, GA) is widely distributed in nature, being a major phenolic pollutant and a commonly used antioxidant and building‐block for drug development. We have characterized the first complete cluster (gal genes) responsible for growth in GA in a derivative of the model bacterium Pseudomonas putida KT2440. GalT mediates specific GA uptake and chemotaxis, and highlights the critical role of GA transport in bacterial adaptation to GA consumption. The proposed GA degradation via the central intermediate 4‐oxalomesaconic acid (OMA) was revisited and all enzymes involved have been identified. Thus, GalD is the prototype of a new subfamily of isomerases that catalyses a biochemical step that remained unknown, i.e. the tautomerization of the OMAketo generated by the GalA dioxygenase to OMAenol. GalB is the founding member of a new family of zinc‐containing hydratases that converts OMAenol into 4‐carboxy‐4‐hydroxy‐2‐oxoadipic acid (CHA). galC encodes the aldolase catalysing CHA cleavage to pyruvic and oxaloacetic acids. The presence of homologous gal clusters outside the Pseudomonas genus sheds light on the evolution and ecology of the gal genes in GA degraders. The gal genes were used for expanding the metabolic abilities of heterologous hosts towards GA degradation, and for engineering a GA cellular biosensor.

Insights into the interaction of discodermolide and docetaxel with tubulin. Mapping the binding sites of microtubule-stabilizing agents by using an integrated NMR and computational approach.

The binding interactions of two antitumor agents that target the paclitaxel site, docetaxel and discodermolide, to unassembled α/β-tubulin heterodimers and microtubules have been studied using biochemical and NMR techniques. The use of discodermolide as a water-soluble paclitaxel biomimetic and extensive NMR experiments allowed the detection of binding of microtubule-stabilizing agents to unassembled tubulin α/β-heterodimers. The bioactive 3D structures of docetaxel and discodermolide bound to α/β-heterodimers were elucidated and compared to those bound to microtubules, where subtle changes in the conformations of docetaxel in its different bound states were evident. Moreover, the combination of experimental TR-NOE and STD NMR data with CORCEMA-ST calculations indicate that docetaxel and discodermolide target an additional binding site at the pore of the microtubules, which is different from the internal binding site at the lumen previously determined by electron crystallography. Binding to this pore site can then be considered as the first ligand-protein recognition event that takes place in advance of the drug internalization process and interaction with the lumen of the microtubules.

Hevein Domains: An Attractive Model to Study Carbohydrate–Protein Interactions at Atomic Resolution

Publisher Summary This chapter focuses on hevein domains and presents an attractive model to study carbohydrate–protein interactions at atomic resolution. Among the various biological processes in which carbohydrates are involved as biochemical signals, it is noteworthy that many plants harbor defense proteins (lectins) against pathogenic attack. These proteins are able to bind to chitin. This natural biopolymer is a key structural component of the cell wall of fungi and of the exoskeleton of invertebrates such as insects, nematodes, and arthropods. Direct binding to the saccharide can occur for the respective lectin, while a particular domain can also be instrumental for chitin-degrading enzymes. The antifungal activity of plant chitinases is largely restricted to those chitinases that contain a noncatalytic, plant-specific, chitin-binding domain (ChBD), also termed as “hevein domain.” This domain displays a common structural motif of 30–43 residues, rich in glycine and cysteine residues in highly conserved positions and organized around a four-disulfide core. The chapter explains the concepts related to protein–carbohydrate interactions and elaborates the basic techniques for analyzing sugar–hevein interactions. It also discusses the structure of the Hevein–Saccharide complexes.

Heparin modulates the mitogenic activity of fibroblast growth factor by inducing dimerization of its receptor. a 3D view by using NMR.

In vitro mitogenesis assays have shown that sulfated glycosaminoglycans (GAGs; heparin and heparan sulfate) cause an enhancement of the mitogenic activity of fibroblast growth factors (FGFs). Herein, we report that the simultaneous presence of FGF and the GAG is not an essential requisite for this event to take place. Indeed, preincubation with heparin (just before FGF addition) of cells lacking heparan sulfate produced an enhancing effect equivalent to that observed when the GAG and the protein are simultaneously added. A first structural characterization of this effect by analytical ultracentrifugation of a soluble preparation of the heparin‐binding domain of fibroblast growth factor receptor 2 (FGFR2) and a low molecular weight (3 kDa) heparin showed that the GAG induces dimerization of FGFR2. To derive a high resolution structural picture of this molecular recognition process, the interactions of a soluble heparin‐binding domain of FGFR2 with two different homogeneous, synthetic, and mitogenically active sulfated GAGs were analyzed by NMR spectroscopy. These studies, assisted by docking protocols and molecular dynamics simulations, have demonstrated that the interactions of these GAGs with the soluble heparin‐binding domain of FGFR induces formation of an FGFR dimer; its architecture is equivalent to that in one of the two distinct crystallographic structures of FGFR in complex with both heparin and FGF1. This preformation of the FGFR dimer (with similar topology to that of the signaling complex) should favor incorporation of the FGF component to form the final assemblage of the signaling complex, without major entropy penalty. This cascade of events is probably at the heart of the observed activating effect of heparin in FGF‐driven mitogenesis.

Dextrans produced by lactic acid bacteria exhibit antiviral and immunomodulatory activity against salmonid viruses

Viral infections in the aquaculture of salmonids can lead to high mortality and substantial economic losses. Thus, there is industrial interest in new molecules active against these viruses. Here we describe the production, purification, and the physicochemical and structural characterization of high molecular weight dextrans synthesized by Lactobacillus sakei MN1 and Leuconostoc mesenteroides RTF10. The purified dextrans, and commercial dextrans with molecular weights ranging from 10 to 2000kDa, were assayed in infected BF-2 and EPC fish cell-line monolayers for antiviral activity. Only T2000 and dextrans from MN1 and RTF10 had significant antiviral activity. This was similar to results obtained against infectious pancreatic necrosis virus. However the dextran from MN1 showed ten-fold higher activity against hematopoietic necrosis virus than T2000. In vivo assays using the MN1 polymer confirmed the in vitro results and revealed immunomodulatory activity. These results together with the high levels of dextran production (2gL(-1)) by Lb. sakei MN1, indicate the compounds potential utility as an antiviral agent in aquaculture.