Louis Patrick Conway

Functional analysis of anomeric sugar kinases

Anomeric sugar kinases perform fundamental roles in the metabolism of carbohydrates. Under- or overexpression of these enzymes, or mutations causing functional impairments can give rise to diseases such as galactosaemia and so the study of this class of kinase is of critical importance. In addition, anomeric sugar kinases which are naturally promiscuous, or have been artificially made so, may find application in the synthesis of libraries of drug candidates (for example, antibiotics), and natural or unnatural oligosaccharides and glycoconjugates. In this review, we provide an overview of the biological functions of these enzymes, the tools which have been developed to investigate them, and the current frontiers in their study.

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.

One assay for all: exploring small molecule phosphorylation using amylose-polyiodide complexes.

We present a generic method for screening small molecule kinases for their acceptor specificity. The release of the reaction byproduct adenosine diphosphate (ADP) triggers a concentration-dependent formation of amylose from sucrose, by using the combined enzymatic action of sucrose synthase and glycogen synthase. Kinase activities could be quantified photometrically after the formation of a dark-blue amylose-polyiodide complex. We demonstrate that this method can be used to profile both known and novel nucleotide- and sugar-kinases for their substrate specificity. Using a facile and widely available methodology, the amylose-polyiodide small-molecule kinase assay presented herein has the potential to perform substrate screenings of small molecule kinases in a high-throughput manner.

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.

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.

Applications of a highly α2,6-selective pseudosialidase

Within human biology, combinations of regioisomeric motifs of α2,6- or α2,3-sialic acids linked to galactose are frequently observed attached to glycoconjugates. These include glycoproteins and glycolipids, with each linkage carrying distinct biological information and function. Microbial linkage-specific sialidases have become important tools for studying the role of these sialosides in complex biological settings, as well as being used as biocatalysts for glycoengineering. However, currently, there is no α2,6-specific sialidase available. This gap has been addressed herein by exploiting the ability of a Photobacterium sp. α2,6-sialyltransferase to catalyze trans-sialidation reversibly and in a highly linkage-specific manner, acting as a pseudosialidase in the presence of cytidine monophosphate. Selective, near quantitative removal of α2,6-linked sialic acids was achieved from a wide range of sialosides including small molecules conjugates, simple glycan, glycopeptide and finally complex glycoprotein including both linkages.

1,3-Di(2-dipyridyl)propan-1,3-dione – a new fluorogenic labeling reagent for milk oligosaccharides

Abstract: We herein demonstrate the use of 1,3-di(2-dipyridyl)propan-1,3-dione (DPPD) as a fluorogenic label for oligosaccharides. A number of milk-derived oligosaccharide standards were successfully labeled with this reagent, with the advantage of greatly simplified sample preparation compared to other commonly used fluorescent tags. DPPD shows a selectivity for oligosaccharides which do not possess a 2-acetamido-2-deoxy-hexose moiety at the reducing terminus, potentially aiding in the identification of complex mixtures of carbohydrates. The use of DPPD for the structural determination of oligosaccharides through exoglycosidase treatment, quantitative analysis of reactions, and in the synthesis of labeled oligosaccharides was also explored. This reagent has, in addition to the analysis of individual and mixed oligosaccharides, potential applications in the study of glycosidases and glycosyltransferases and as such represents a valuable addition to the tools available to the glycoscientist.

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.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.