C-Terminal Truncation of α 1,6-Fucosyltransferase from Rhizobium Sp. does not Annul the Transferase Activity of the Enzyme

6 pages.-- PMID: 11814863 [PubMed].Recently we have over-expressed the enzyme a 1,6-fucosyltransferase from Rhizobium sp. in Escherichia coli. In this heterologous system the enzyme was mainly expressed as inclusion bodies and the one that was expressed soluble showed a shortlasting activity in solution due to precipitation of the protein. A structural analysis of the sequence using the TMpred program predicted a highly hydrophobic region of 19 aa close to the C-terminal of the protein. In order to investigate the influence of this region on the formation of inclusion bodies and the precipitation from solution, we cloned a truncated version of the protein where a C-terminal fragment of 65 aa, including the predicted transmembrane-like region, was removed. The resulting protein was expressed in a soluble form without formation of inclusion bodies. The truncated protein catalyzed the transfer of a fucopyranosyl moiety from GDP-b-l-Fucose to chitobiose. Comparison of the acceptor specificity between the truncated a 1,6-fucosyltransferase and the wild-type enzyme, showed a similar behavior for both enzymes. Our results indicate that the active center is not located in the C-terminal extreme of the protein in contrast to the case of the mammalian glycosyltransferases. Also, these results indicate that the a-6-motif III is not directly involved in the catalytic activity of the enzyme.This work was supported by the Spanish DGES (Grant PB96-0828) and Comunidad de Madrid (Grant 07B/0027/1999).Peer reviewe

6-O-Nucleotidyltransferase: an aminoglycoside-modifying enzyme specific for streptomycin/streptidine

Aminoglycosides are especially useful for the treatment of hospital-acquired infections. The main problem for the application of these antibiotics is the presence of bacterial resistance enzymes, in particular, nucleotidyltransferases (ANTs). These enzymes catalyze the transfer of an adenylyl group from the MgATP complex to different positions of the antibiotic. To understand the mechanisms that lead to antibiotic inactivation, we have performed a comprehensive experimental analysis of one of those enzymes. The 6-O- nucleotidyltransferase enzyme (ANT(6)) from Bacillus subtilis was cloned, overexpressed and purified in E. coli. The kinetic parameters revealed a narrow specificity of the ANT(6) for MgATP/streptomycin as substrates. The binding epitope of the streptomycin recognized by the ANT(6) is the streptidine moiety. Therefore, the use of streptidine as a “decoy acceptor” allows the recovery of the antibiotic activity of streptomycin E. coli cells that are overexpressing the ANT(6).This investigation was supported by a research grant of the Span-ish research “Dirección General de Investigación" CTQ2013-45538-PPeer reviewe

Theoretical Studies on Mechanism of Inactivation of Kanamycin A by 4′-O-Nucleotidyltransferase

This work is focused on mechanistic studies of the transfer of an adenylyl group (Adenoside-5′-monophosfate) from adenosine 5′-triphosphate (ATP) to a OH-4′ hydroxyl group of an antibiotic. Using hybrid quantum mechanics/molecular mechanics (QM/MM) techniques, we study the substrate and base-assisted mechanisms of the inactivation process of kanamycin A (KAN) catalyzed by 4′-O-Nucleotidyltransferase [ANT(4′)], an active enzyme against almost all aminoglycoside antibiotics. Free energy surfaces, obtained with Free Energy Perturbation methods at the M06-2X/MM level of theory, show that the most favorable reaction path presents a barrier of 12.2 kcal·mol−1 that corresponds to the concerted activation of O4′ from KAN by Glu145. In addition, the primary and secondary 18O kinetic isotope effects (KIEs) have been computed for bridge O3α, and non-bridge O1α, O2α, and O5′ atoms of ATP. The observed normal 1°-KIE of 1.2% and 2°-KIE of 0.07% for the Glu145-assisted mechanism are in very good agreement with experimentally measured data. Additionally, based on the obtained results, the role of electrostatic and compression effects in enzymatic catalysis is discussed

Interfering with mRNA methylation by the 2′O-Methyltransferase (NSP16) from SARS-CoV-2 to tackle the COVID-19 disease

The pandemic associated to Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) has resulted in a huge number of deaths and infected people. Although several vaccine programmes are currently underway and have reached phase 3, and a few small size drugs repurposed to aid treatment of severe cases of COVID-19 infections, effective therapeutic options for this disease do not currently exist. NSP16 is a S-adenosyl-L-Methionine (SAM) dependent 2′O-Methyltransferase that converts mRNA cap-0 into cap-1 structure to prevent virus detection by cell innate immunity mechanisms. NSP16 methylates the ribose 2′O-position of the first nucleotide of the mRNA only in the presence of an interacting partner, the protein NSP10. This feature suggests that inhibition of the NSP16 may represent a therapeutic window to treat COVID-19. To test this idea, we performed comparative structural analyses of the NSP16 present in human coronaviruses and developed a sinefungin (SFG) similarity-based virtual screening campaign to assess the druggability of the SARS-CoV-2 NSP16 enzyme. Through these studies, we identified the SFG analogue 44601604 as a promising more potent inhibitor of NSP16 to limit viral replication in infected cells, favouring viral clearance

Preparation and Characterization of Aminoglycoside-Loaded Chitosan/Tripolyphosphate/Alginate Microspheres against E. coli

Although aminoglycosides are one of the common classes of antibiotics that have been widely used for treating infections caused by pathogenic bacteria, the evolution of bacterial resistance mechanisms and their inherent toxicity have diminished their applicability. Biocompatible carrier systems can help sustain and control the delivery of antibacterial compounds while reducing the chances of antibacterial resistance or accumulation in unwanted tissues. In this study, novel chitosan gel beads were synthesized by a double ionic co-crosslinking mechanism. Tripolyphosphate and alginate, a polysaccharide obtained from marine brown algae, were employed as ionic cross-linkers to prepare the chitosan-based networks of gel beads. The in vitro release of streptomycin and kanamycin A was bimodal; an initial burst release was observed followed by a diffusion mediated sustained release, based on a Fickian diffusion mechanism. Finally, in terms of antibacterial properties, the particles resulted in growth inhibition of Gram-negative (E. coli) bacteria

Identification of novel potential heparanase inhibitors using virtual screening

Heparanase (HPSE) is a mammalian endo-β-D-glucuronidase that cleaves heparan sulphate (HS) side chains of heparin sulphate proteoglycans (HSPG), a class of molecules composed of repeating polysulfated disaccharide units of glucosamine and hexuronic acid residues. HPSE controls the availability of growth factors, chemokines, lipoproteins and other bioactive molecules by degrading HS into smaller fractions, allowing the release of saccharide fragments that activate a plethora of signaling processes. HPSE overexpression has been correlated with tumor survival and metastasis as well as several diseases associated with chronic inflammation, including the ongoing COVID-19 pandemic caused by SARS-CoV-2. Thus, the search for molecules that could potentially inhibit HPSE has become increasingly relevant in the clinic. In this study, we have integrated a strategy that combines virtual screening and molecular docking of publicly available chemical databases to identify small compounds that can be developed into novel HPSE inhibitors. Structural rationalization of the interactions previously reported compounds led us to identify promising unexplored chemotypes. Here we show that these novel potential HPSE inhibitors present optimized in silico druggability and docking properties and may serve as pharmacological tools for the treatment of chronic and infectious diseases associated with chronic inflammation

A nanostructured Cu(II) coordination polymer based on alanine as a trifunctional mimic enzyme and efficient composite in the detection of Sphingobacteria

This research raises the potential use of coordination polymers as new useful materials in two essential research fields, allowing the obtaining of a new multiartificial enzyme with the capacity to inhibit the growth of bacteria resistance. The fine selection of the ligands allows the design of a new 2D coordination polymer (CP), with the formula [Cu2(IBA)2(OH2)4]n·6nH2O, by the combination of Cu (II) as the metal center with a pseudoamino acid (H2IBA = isophthaloyl bis β-alanine). Quantitative total X-ray fluorescence (TXRF) analyses show that the obtained CP can gradually release Cu (II) ions. Additionally, this CP can be nanoprocessed and transformed into a metal-organic gel (MOG) by using different Cu (II) salt concentrations and the application of ultrasounds. Considering its nanometric dimensions, the slow Cu (II) release and its simple processability, its performance as an artificial enzyme, and its antibacterial ability were explored. The results obtained show the first nanocoordination polymer acting as an artificial multienzyme (peroxidase, catalase, and superoxodismutase) exhibiting antibacterial activity in the presence of hydrogen peroxide, with selective behavior for three bacterium strains (S. spiritovirum, A. faecales, and B. cereus). Indeed, this CP shows a more robust inhibition capacity for Sphingobacterium. Going beyond that, as there are no comfortable and practically clinical tests capable of detecting the presence of Sphingobacteria, the compound can be easily embedded to form moldable gelatin that will facilitate the handling and low-cost commercial kit

Chemoenzymatic preparation of Chondroitin sulfates with a defined sulfation pattern

Comunicación Oral impartida en el XVII European Congress on BiotechnologyN