Elucidating paramylon and other carbohydrate metabolism in Euglena gracilis: Kinetic characterization, structure and cellular localization of UDP-glucose pyrophosphorylase (original) (raw)
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Structure-function studies of carbohydrate-active enzymes
Carbohydrate Bioengineering
Carbohydrate-active enzymes are responsible for both the biosynthesis and breakdown of carbohydrates and glycoconjugates. They are involved in many metabolic pathways, in the biosynthesis and degradation of various biomolecules, such as bacterial exopolysaccharides, starch, cellulose, and lignin, and in the glycosylation of proteins and lipids. Their interesting reactions have attracted the attention of researchers belonging to different scientific fields ranging from basic research to biotechnology. Interest in carbohydrate-active enzymes is due not only to their ability to build and degrade biopolymers, which is highly relevant in biotechnology, but also because they are involved in bacterial biofilm formation, and in the glycosylation of proteins and lipids, which has important health implications. This Special Issue features a collection of research papers and reviews representing an up-to-date state of the art in this growing field of research to broaden our understanding about carbohydrate-active enzymes, their mutants, and their reaction products at the molecular level. Tindara Venuto et al. wrote a paper about alpha2,8-sialyltransferases from ray-finned fish and showed that, in tetrapods, duplicated st8sia genes like st8sia7, st8sia8, and st8sia9 have disappeared while orthologues are maintained in teleosts [1]. They reconstructed the evolutionary history of st8sia genes in fish genomes and by bioinformatics analysis showed changes in the conserved polysialyltransferase domain of the fish sequences possibly accounting for variable enzymatic activities [1]. PhNah20A, a β-N-acetylhexosaminidase from the marine bacterium Paraglaciecola hydrolytica S66T, was identified and characterized. By phylogenetic analysis, the authors discovered that PhNah20A is located outside clusters of other studied β-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. PhNah20A is the first characterized member of a distinct subgroup within GH20 β-N-acetylhexosaminidases. The recombinant PhNah20A showed optimum hydrolytic activity on GlcNAc β-1,4 and β-1,3 linkages in chitobiose (GlcNAc)2 and GlcNAc-1,3-β-Gal-1,4-β-Glc (LNT2), the core structure of a human milk oligosaccharide, at pH 6.0 and 50 • C. Interestingly PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide β-Gal-1,4-β-Glc-1,1-β-GlcNAc, and in low amounts the β-1,2or β-1,3-linked trisaccharide β-Gal-1,4(β-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor [2]. The production of biofuels from lignocellulosic biomasses is hampered by the saccharification step of the biomasses requiring the concerted action of lignocellulose cellulases and hemicellulases. Zhang et al. characterized the secretome of Trichoderma harzianum EM0925 under induction of lignocellulose cellulases and hemicellulases [3]. Compared with the commercial enzyme preparations, the T. harzianum EM0925 enzyme cocktail presented significantly higher lignocellulolytic enzyme activities and hydrolysis efficiency against lignocellulosic biomasses. The enzyme mixture used on ultrafine grinding and alkali pretreated corn stover yielded 100% glucose and xylose. The authors suggest that natural cellulases mixed with the hemicellulases enzyme cocktail from T. harzianum
Genomic or cDNA clones for the glycolytic enzyme enolase were isolated from the amitochondriate pelobiont Mastigamoeba balamuthi, from the kinetoplastid Trypanosoma brucei, and from the euglenid Euglena gracilis. Clones for the cytosolic enzyme were found in all three organisms, whereas Euglena was found to also express mRNA for a second isoenzyme that possesses a putative N-terminal plastid-targeting peptide and is probably targeted to the chloroplast. Database searching revealed that Arabidopsis also possesses a second enolase gene that encodes an N-terminal extension and is likely targeted to the chloroplast. A phylogeny of enolase amino acid sequences from 6 archaebacteria, 24 eubacteria, and 32 eukaryotes showed that the Mastigamoeba enolase tended to branch with its homologs from Trypanosoma and from the amitochondriate protist Entamoeba histolytica. The compartment-specific isoenzymes in Euglena arose through a gene duplication independent of that which gave rise to the compartment-specific isoenzymes in Arabidopsis, as evidenced by the finding that the Euglena enolases are more similar to the homolog from the eubacterium Treponema pallidum than they are to homologs from any other organism sampled. In marked contrast to all other glycolytic enzymes studied to date, enolases from all eukaryotes surveyed here (except Euglena) are not markedly more similar to eubacterial than to archaebacterial homologs. An intriguing indel shared by enolase from eukaryotes, from the archaebacterium Methanococcus jannaschii, and from the eubacterium Campylobacter jejuni maps to the surface of the three-dimensional structure of the enzyme and appears to have occurred at the same position in parallel in independent lineages.
Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC, HPLC and mass spectrometry, proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial membranes, as well as α- and β-GalT activities in trypanosomal membranes, benchmarking the potential of the fluorescent acceptor approach against earlier radiochemical assays. Following on to explore the glycobiology of the benign protozoan alga Euglena gracilis, α-1,3- and α-1,2-ManT activities were detected in membrane preparations, along with GlcT, Glc-P-T and GlcNAc-P-T activities. These studies serve to demonstrate the potential of readily accessible fluorescent glycans as substrates for exploring carbohydrate active enzymes.
Bioorganic & medicinal chemistry, 1996
Two enzymes of the Leloir pathway, UDP-GlcNAc pyrophosphorylase and UDP-Glc dehydrogenase, which are involved in the synthesis of activated sugar nucleotides have been cloned, overexpressed in Escherichia coli, and purified to homogeneity in only one step by chelation-affinity chromatography. The gene KfaC of E. coli K5 was thus demonstrated to encode UDP-Glc DH. Some properties of the cloned enzymes, such as stability, pH dependence, and substrate kinetics, were studied in order to facilitate the use of these enzymes in carbohydrate synthesis, especially in the synthesis of hyaluronic acid.
Domain Swapping between a Cyanobacterial and a Plant Subunit ADP-Glucose Pyrophosphorylase
Plant and Cell Physiology, 2006
ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the regulatory step in the pathway for synthesis of bacterial glycogen and starch in plants. ADP-Glc PPases from cyanobacteria (homotetramer) and from potato (Solanum tuberosum) tuber (heterotetramer) are activated by 3-phosphoglycerate and inhibited by inorganic orthophosphate. To study the function of two putative domains, chimeric enzymes were constructed. PSSANA contained the N-terminus (292 amino acids) of the potato tuber ADP-Glc PPase small subunit (PSS) and the C-terminus (159 residues) of the Anabaena PCC 7120 enzyme. ANAPSS was the inverse chimera. These constructs were expressed separately or together with the large subunit of the potato tuber ADP-Glc PPase (PLS), to obtain homo-and heterotetrameric chimeric proteins. Characterization of these forms showed that the N-terminus determines stability and regulatory redox-dependent properties. The chimeric forms exhibited intermediate 3-phosphoglycerate activation properties with respect to the wild-type homotetrameric enzymes, indicating that the interaction between the putative N-and C-domains determines the affinity for the activator. Characterization of the chimeric heterotetramers showed the functionality of the large subunit, mainly in modulating regulation of the enzyme by the coordinate action of 3phosphoglycerate and inorganic orthophosphate.
Open and Closed Structures of the UDP-glucose Pyrophosphorylase from Leishmania major
Journal of Biological Chemistry, 2007
Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel -helix (LH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.
PLANT PHYSIOLOGY, 1989
The membrane-bound UDP-glucose-fl-(1,3)-glucan synthase from Daucus carota L. was characterized and a solubilization procedure was developed. The enzyme exhibited maximal activity in the presence of 0.75 millimolar Ca2", 0.5 millimolar EGTA, and 5 millimolar cellobiose at pH 7.5 and 30°C at 1 millimolar UDPG. Reaction products were confirmed to be (1,3)-linked glucan. Polypeptides of 150, 57, and 43 kilodaltons were labeled with the photoactivatible affinity label 5-azido-uridine 5'-iO_[32p] diphosphateglucose. Labeling of the 150 and 57 kilodalton polypeptides was completely protected against by 1 millimolar nonradioactive UDPG suggesting that one or both of these polypeptides may represent the UDPG binding subunit of glucan synthase. Carrot glucan synthase was solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in the absence of divalent cations and chelators; however, the percentage of enzyme which could be solubilized showed variability with membrane source. With microsomal membranes, up to 80% of the enzyme was released with 0.7% CHAPS. Solubilized enzyme was stable for at least 9 hours at 40C. When more highly purified membrane fractions were isolated from sucrose step gradients a slightly different picture emerged. Activity from the 20/30% interface (Golgi and tonoplast enriched) was readily solubilized and expressed. Activity from the 30/40% interface (plasma membrane enriched) was also solubilized; however, it was necessary to add heat inactivated microsomes to assay mixtures for full activity to be expressed. A requirement for endogenous activators is suggested.