The Shewanella woodyi galactokinase pool phosphorylates glucose at the 6-position.

Galactokinases are a class of enzymes which belong to the GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) superfamily and catalyse the phosphorylation of galactose in the first step of the Leloir pathway. Here we report the discovery of three enzymes from Shewanella woodyi which have been classified as galactokinases based on sequence similarity. However, each of these enzymes show little to no significant activity towards galactose and instead exhibit a strong preference for glucose. Furthermore, in contrast to the usual galactose-1-phosphate product of the galactokinase-catalysed reaction, these enzymes produce glucose-6-phosphate. This radical change in enzyme functionality is postulated to be linked to the mutation of a glycine residue which is conserved in all other sequenced galactokinases.

Discovery and Biochemical Characterization of UDP-Glucose Dehydrogenase from Akkermansia muciniphila.

BACKGROUND: The biocatalytic oxidation of UDP-glucose in the presence of NAD+ is catalyzed by UDP-glucose dehydrogenases. OBJECTIVES: The main objective of this study was the characterization of a UDP-glucose dehydrogenase (AmUGD) from Akkermansia muciniphila, a bacterium originally isolated from human faeces in an anaerobic medium containing gastric mucin as the sole carbon source. METHODS: The biochemical analysis of AmUGD was performed using a plate reader-based assay measuring the reaction by-product NADH. Furthermore, HPLC- and MALDI-ToF-MS- based methods were used for the enzyme characterization. RESULTS: The recombinant form of the protein was expressed in E. coli and the purified enzyme exhibited optimum levels of activity at 37°C and pH 9.0. While the enzyme is active in the absence of metal ions, the presence of Zn2+ ions results in markedly enhanced levels of catalysis. CONCLUSION: This study describes the first characterization of a nucleotide-processing enzyme from A. muciniphila. The ease of expression and purification of this enzyme make it ideal for biotechnological applications such as the enzymatic synthesis of nucleotide sugars, which may in turn be used for the synthesis of complex carbohydrates or glycoconjugates.

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.