Biochemical characterization of the endo-α-N-acetylgalactosaminidase pool of the human gut symbiont Tyzzerella nexilis.

Three large (2084-, 984-, and 2104-amino acids) endo-α-N-acetylgalactosaminidase candidate genes from the commensal human gut bacterium Tyzzerella nexilis were successfully cloned and subsequently expressed in Escherichia coli. Activity tests of the purified proteins revealed that two of the candidate genes (Tn0153 and Tn2105) were able to hydrolyze the disaccharide unit from Galß1-3GalNAc-α-pNP. The biochemical characterization revealed optimum pH conditions of 4.0 for both enzymes and temperature optima of 50 °C. The addition of 2-mercaptoethanol, Triton X-100 and urea had only minor effects on the activity of the enzymes, and the addition of imidazole and sodium dodecyl sulfate led to a significant reduction of the enzymes' activities. A mutational study identified and confirmed the role of the catalytically significant amino acids. The present study describes the first functional characterization of members of the GH101 family from this human gut symbiont.

Pseudomonas putida Represses JA- and SA-Mediated Defense Pathways in Rice and Promotes an Alternative Defense Mechanism Possibly through ABA Signaling.

The signaling pathways induced by Pseudomonas putida in rice plants at the early plant-rhizobacteria interaction stages, with and without inoculation of Xanthomonas oryzae pv. oryzae, were studied. In the absence of pathogen, P. putida reduced ethylene (ET) production, and promoted root and stem elongation. Interestingly, gene OsHDA702, which plays an important role in root formation, was found significantly up-regulated in the presence of the rhizobacterium. Although X. oryzae pv. oryzae inoculation enhanced ET production in rice plants, P. putida treatment repressed ET-, jasmonic acid (JA)- and salicylic acid (SA)-mediated defense pathways, and induced the biosynthesis of abscisic acid (ABA), and the overexpression of OsHDA705 and some pathogenesis-related proteins (PRs), which in turn increased the susceptibility of the rice plants against the pathogen. Collectively, this is the first work on the defense signaling induced by plant growth-promoting rhizobacteria in plants at the early interaction stages, and suggests that rhizobacteria stimulate an alternative defense mechanism in plants based on ABA accumulation and OsHDA705 signaling.

Effects of microvirin monomers and oligomers on hepatitis C virus.

Microvirin (MVN) is a carbohydrate-binding protein which shows high specificity for high-mannose type N-glycan structures. In the present study, we tried to identify whether MVN could bind to high-mannose containing hepatitis C virus (HCV) envelope glycoproteins, which are heavily decorated high-mannose glycans. In addition, recombinantly expressed MVN oligomers in di-, tri- and tetrameric form were evaluated for their viral inhibition. MVN oligomers bound more efficiently to HCV virions, and displayed in comparison with the MVN monomer a higher neutralization potency against HCV infection. The antiviral effect was furthermore affected by the peptide linker sequence connecting the MVN monomers. The results indicate that MVN oligomers such as trimers and tetramers may be used as future neutralization agents against HCV infections.

Discovery and Biochemical Characterization of a Thermostable Glucose-1-phosphate Nucleotidyltransferase from Thermodesulfatator indicus.

BACKGROUND: The biosynthesis of NDP-glucoses is based on the nucleotide transfer from NTP donor substrates to glucose-1-phosphates catalyzed by glucose-1-phosphate nucleotidyltransferases. OBJECTIVES: The cloning and biochemical characterization of a glucose-1-phosphate nucleotidyltransferase (TiGPNT) from the deep sea bacterium Thermodesulfatator indicus. METHODS: The biochemical parameters of recombinant TiGPNT were determined using a plate reader-based coupled enzymatic assay, in which the reaction product UDP-glucose is oxidized in the presence of NAD+ forming UDP-Glucuronic acid and NADH. The substrate promiscuity of the enzyme was determined using thin-layer chromatography and MALDI-ToF mass spectrometry. RESULTS: TiGPNT was recombinantly expressed under the control of the T7 promoter in Escherichia coli and could be successfully enriched by heat treatment at 80°C for 30 min. The obtained enzyme worked best at pH 7.5 and the optimum reaction temperature was determined to be 50°C. Interestingly, TiGPNT could fully retain its activity even after extended incubation periods at temperatures of up to 80°C. The enzyme was strongly inhibited in the presence of Cu2+ and Fe2+ ions and EDTA. Among the tested glycosyl donor substrates, TiGPNT showed strict specificity towards glucose-1-phosphate. At the same time, TiGPNT was highly promiscuous towards all tested nucleotide donor substrates. CONCLUSION: TiGPNT shows comparable biochemical features in regards to pH optima, temperature optima and the substrate specificity to characterized glucose-1-phosphate nucleotidyltransferase from other species. The enzyme was capable of utilizing glucose-1-phosphate and all tested nucleoside triphosphate donors as substrates. The high activity of the enzyme and the simple purification protocol make TiGPNT an interesting new biocatalyst for the synthesis of glucose-diphospho nucleosides.

An integrated 3D-printed platform for the automated isolation of N-glycans.

The development of techniques for the rapid analysis of N-glycans is a key step in enabling the roles of glycoproteins in biological processes to be studied. Analysis is usually performed through the liberation of the carbohydrate moieties from proteins, followed by fluorescent labeling and identification using either standardized HPLC or mass spectrometry techniques. A simple and robust automated process for the release and isolation of N-glycans would greatly improve analytical throughput and reproducibility, and is thus highly desirable. Inspired by the increasing number of reported projects involving open source labware, which allows the design and construction of otherwise inaccessible laboratory equipment using low-cost 3D printers, we used this technique to fabricate a platform for the automated isolation of N-glycans. As a proof of concept, we demonstrated the successful recovery of glycan samples from the glycoprotein model fetuin using our self-made 3D-printed equipment.

Discovery and Biochemical Characterization of the UDP-Xylose Biosynthesis Pathway in Sphaerobacter thermophilus.

The biosynthesis of UDP-xylose requires the stepwise oxidation/ decarboxylation of UDP-glucose, which is catalyzed by the enzymes UDPglucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS). UDPxylose biosynthesis is ubiquitous in animals and plants. However, only a few UGD and UXS isoforms of bacterial origin have thus far been biochemically characterized. Sphaerobacter thermophilus DSM 20745 is a bacterium isolated from heated sewage sludge, and therefore can be a valuable source of thermostable enzymes of biotechnological interest. However, no biochemical characterizations of any S. thermophilus enzymes have yet been reported. Herein, we describe the cloning and characterization of putative UGD (StUGD) and UXS (StUXS) isoforms from this organism. HPLC- and plate reader-based activity tests of the recombinantly expressed StUGD and StUXS showed that they are indeed active enzymes. Both StUGD and StUXS showed a temperature optimum of 70°C, and a reasonable thermal stability up to 60°C. No metal ions were required for enzymatic activities. StUGD had a higher pH optimum than StUXS. The simple purification procedures and the thermotolerance of StUGD and StUXS make them valuable biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose at elevated temperatures. The biosynthetic potential of StUGD was further exemplified in a coupled enzymatic reaction with an UDP-glucuronosyltransferase, allowing the glucuronylation of the natural model substrate bilirubin.