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

Exploring the Origin of Amidase Substrate Promiscuity in CALB by a Computational Approach

Enzyme promiscuity attracts the interest of the industrial and academic sectors because of its application in the design of biocatalysts. The amidase activity of Candida antarctica lipase B (CALB) on two different substrates has been studied by theoretical quantum mechanics/molecular mechanics methods, supported by experimental kinetic measurements. The aim of the study is to understand the substrate promiscuity of CALB in this secondary reaction and the origin of its promiscuous catalytic activity. The computational results predict activation free energies in very good agreement with the kinetic data and confirm that the activity of CALB as an amidase, despite depending on the features of the amide substrate, is dictated by the electrostatic effects of the protein. The protein polarizes and activates the substrate as well as stabilizes the transition state, thus enhancing the rate constant. Our results can provide guides for future designs of biocatalysts based on electrostatic arguments

Development of a new method for D-xylose detection and quantification in urine, based on the use of recombinant xylose dehydrogenase from Caulobacter crescentus.

The gene xylB from Caulobacter crescentus has been cloned and expressed in Escherichia coli providing a high yield of xylose dehydrogenase (XylB) production and excellent purity (97%). Purified recombinant XylB showed an absolute dependence on the cofactor NAD+ and a strong preference for d-xylose against other assayed mono and disaccharides. Additionally, XylB showed strong stability when stored as freeze-dried powder at least 250 days both at 4 °C and room temperature. In addition, more than 80% of the initial activity of rehydrated freeze-dried enzyme remained after 150 days of incubation at 4 °C. Based on these characteristics, the capability of XylB in d-xylose detection and quantification was studied. The linearity of the method was maintained up to concentrations of d-xylose of 10 mg/dL and the calculated limits of detection (LoD) and quantification (LoQ) of xylose in buffer were 0.568 mg/dL and 1.89 mg/dL respectively. Thus, enzymatic detection was found to be an excellent method for quantification of d-xylose in both buffer and urine samples. This method can easily be incorporated in a new test for the diagnosis of hypolactasia through the measurement of intestinal lactase activity.This work was supported by a grant from Venter Pharma SL (Spain) and partially supported by the Spanish Ministerio de Economía y Competitividad (Grant MAT2015-65184-C2-2-R, MINECO/FEDER).Peer reviewe

Computational Study of the Phosphoryl Donor Activity of Dihydroxyacetone Kinase from ATP to Inorganic Polyphosphate

Adenosine triphosphate (ATP) is the main biological phosphoryl donor required in many enzymes including dihydroxyacetone kinases (DHAKs) that convert dihydroxyacetone (Dha) into dihydroxyacetone phosphate (Dha-P), a key species with potential applications in synthesis. Herein, we present a theoretical study of the molecular mechanism for the phosphoryl transfer reaction from an inorganic polyphosphate to Dha catalyzed by DHAK from C. freundii. This is part of a project devoted to modify the phosphoryl donor specificity of this enzyme avoiding the use of the problematic direct addition of ATP. Based on the use of hybrid QM/MM potentials, with the QM region described by semiempirical and DFT methods, the reaction mechanism of the wild-type enzyme and the most active experimentally measured mutant (Glu526Lys) with poly-P as phosphoryl donor has been explored to elucidate the origin of the activity of this mutant. The similar energy barriers obtained in both systems confirm our previous studies on the binding step (Sánchez-Moreno et al., Int. J. Mol. Sci. 2015, 16, 27835) suggesting that this mutation favors a more adequate position of the poly-P in the active site for the following step, the chemical reaction, to take place

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

Preparation and characterization of a bifunctional aldolase/kinase enzyme. A more efficient biocatalyst for C-C bond formation

“This is the pre-peer reviewed version of the following article: Iturrate, L., Sánchez-Moreno, I., Oroz-Guinea, I., Pérez-Gil, J., García-Junceda, E. (2010) “Preparation and characterization of a bifunctional aldolase/kinase enzyme. A more efficient biocatalyst for C-C bond formation“ Chem. Eur. J., 16, 4018-4030, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1002/chem.200903096/abstract”A bifunctional aldolase/kinase enzyme named DLF has been constructed by gene fusion through overlap extension. This fusion enzyme consists of monomeric fructose-1,6-bisphosphate aldolase (FBPA) from Staphylococcus carnosus and the homodimeric dihydroxyacetone kinase (DHAK) from Citrobacter freundii CECT 4626 with an intervening five amino acid linker. The fusion protein was expressed soluble and retained both kinase and aldolase activities. The secondary structure of the bifuctional enzyme has been analysed by CD spectroscopy, as well as that of the parental enzymes, in order to study the effect of the covalent coupling of the two parent proteins on the structure of the fused enzyme. Since S. carnosus FBPA is a thermostable protein, the effect of the fusion on the thermal stability of the bifunctional enzyme has also been studied. The proximity of the active centres in the fused enzyme promotes a kinetic advantage as the 20-fold increment in the initial velocity of the overall aldol reaction indicates. Experimental evidence supports that this increase in the reaction rate can be explained in terms of substrate channellingWe thank the Spanish Ministerio de Ciencia e Innovación for financial support (Grant CTQ2007-67403/BQU). J. P.-G. has been supported by grants BIO2009-09694 and CSD2007-00010. L. Iturrate and I. Sánchez-Moreno acknowledges the Predoctoral Fellowship from Comunidad de Madrid. I. Oroz-Guinea is a JAEPredoc fellow from CSIC. We thank E. G. Doyagüez for her assistance on NMR characterization

Design of a biocatalytic cascade for the enzymatic sulfation of unsulfated chondroitin with in situ generation of PAPS

Sulfation of molecules in living organisms is a process that plays a key role in their functionality. In mammals, the sulfation of polysaccharides (glycosaminoglycans) that form the proteoglycans present in the extracellular matrix is particularly important. These polysaccharides, through their degree and sulfation pattern, are involved in a variety of biological events as signal modulators in communication processes between the cell and its environment. Because of this great biological importance, there is a growing interest in the development of efficient and sustainable sulfation processes, such as those based on the use of sulfotransferase enzymes. These enzymes have the disadvantage of being 3′-phosphoadenosine 5′-phosphosulfate (PAPS) dependent, which is expensive and difficult to obtain. In the present study, a modular multienzyme system was developed to allow the in situ synthesis of PAPS and its coupling to a chondroitin sulfation system. For this purpose, the bifunctional enzyme PAPS synthase 1 (PAPSS1) from Homo sapiens, which contains the ATP sulfurylase and APS kinase activities in a single protein, and the enzyme chondroitin 4-O-sulfotransferase (C4ST-1) from Rattus norvegicus were overexpressed in E. coli. The product formed after coupling of the PAPS generation system and the chondroitin sulfation module was analyzed by NMR

Chemoenzymatic preparation of Chondroitin sulfates with a defined sulfation pattern

Comunicación Oral impartida en el XVII European Congress on BiotechnologyN

Multi-Step Enzyme Catalysis: Biotransformations and Chemoenzymatic Synthesis

The first comprehensive coverage of this unique and interdisciplinary field provides a complete overview, covering such topics as chemoenzymatic synthesis, microbial production of DNA building blocks, asymmetric transformations by coupled enzymes and much more. By combining enzymatic and synthetic organic steps, the use of multi-enzyme complexes and other techniques opens the door to reactions hitherto unknown, making this monograph of great interest to biochemists, organic chemists, and chemists working with/on organometallics, as well as catalytic chemists, biotechnologists, and those working in the pharmaceutical and fine chemical industries.Front Matter (pages I–XV) Chapter 1 Asymmetric Transformations by Coupled Enzyme and Metal Catalysis: Dynamic Kinetic Resolution (pages 1–19) Mahn-Joo Kim, Jaiwook Park and Yoon Kyung Choi Chapter 2 Chemoenzymatic Routes to Enantiomerically Pure Amino Acids and Amines (pages 21–39) Nicholas J. Turner Chapter 3 Oxidizing Enzymes in Multi-Step Biotransformation Processes (pages 41–60) Stephanie G. Burton and Marilize le Roes-Hill Chapter 4 Dihydroxyacetone Phosphate-Dependent Aldolases in the Core of Multi-Step Processes (pages 61–81) Laura Iturrate and Eduardo García-Junceda Chapter 5 Multi-Enzyme Systems for the Synthesis of Glycoconjugates (pages 83–107) Birgit Sauerzapfe and Lothar Elling Chapter 6 Enzyme-Catalyzed Cascade Reactions (pages 109–135) Roger A. Sheldon Chapter 7 Multi-Modular Synthases as Tools of the Synthetic Chemist (pages 137–158) Michael D. Burkart and Junhua Tao Chapter 8 Modifying the Glycosylation Pattern in Actinomycetes by Combinatorial Biosynthesis (pages 159–198) José A. Salas and Carmen Méndez Chapter 9 Microbial Production of DNA Building Blocks (pages 199–211) Jun Ogawa, Nobuyuki Horinouchi and Sakayu Shimizu Chapter 10 Combination of Biocatalysis and Chemical Catalysis for the Preparation of Pharmaceuticals Through Multi-Step Syntheses (pages 213–233) Vicente Gotor-Fernández, Rosario Brieva and Vicente Gotor SummaryPDF(237K)ReferencesServicio de Enlaces CSICYou have free access to this contentIndex (pages 235–241)Peer reviewe