Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: UNIPROT:Q8NEX9 (reductase)
26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The first committed enzyme in GDP-L-fucose formation from GDP-D-mannose is GDP-D-mannose 4,6-dehydratase, which forms GDP-4-keto-6-deoxy-D-mannose. The uncertain enzymatic steps beyond this point were examined in this study. Assays were developed for the epimerase and reductase activities which the putative pathway would predict. A protein was isolated exhibiting homogeneity by several criteria. This single protein, which forms GDP-L-fucose from GDP-4-keto-6-deoxy-D-mannose and NADH, appears to possess both epimerase and reductase capabilities and may be termed GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase. Analysis on a molecular sieve column using fast protein liquid chromatography established a molecular weight of 63,100 for the native enzyme, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis established a subunit molecular weight of 31,500.
...
PMID:An epimerase-reductase in L-fucose synthesis. 333 88

We have cloned the cDNA encoding human GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose. The message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase activity. The human GDP-mannose 4,6-dehydratase polypeptide shares 61% identity with the enzyme from Escherichia coli, suggesting broad evolutionary conservation. Purified recombinant enzyme utilizes NADP+ as a cofactor and, like its E. coli counterpart, is inhibited by GDP-fucose, suggesting that this aspect of regulation is also conserved. We have isolated the product of the dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure by electrospray ionization-mass spectrometry and high field NMR. Using purified recombinant human GDP-mannose 4,6-dehydratase and FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we show that the two proteins alone are sufficient to convert GDP-mannose to GDP-fucose in vitro. This unequivocally demonstrates that the epimerase and reductase activities are on a single polypeptide. Finally, we show that the two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway in bacteria.
...
PMID:Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro. 952 24

The GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GM_ER) isolated from E. coli has been overexpressed as a GST-fusion protein and purified to homogeneity. The enzyme, an NADP+(H)-binding homodimer of 70 kDa, is responsible for the production of GDP-L-fucose. GM_ER shows significant structural homology to the human erythrocyte protein FX, which is involved in blood-group glycoconjugate biosynthesis, displaying 3,5 epimerase/reductase activity on GDP-4-keto-6-deoxy-D-mannose. GM_ER has been crystallized in a trigonal crystalline form, containing one molecule per asymmetric unit, suitable for high-resolution crystallographic investigations.
...
PMID:Preliminary crystallographic investigations of recombinant GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from E. coli. 976 75

The 6-deoxyhexose L-fucose is an important and characteristic element in glycoconjugates of bacteria (e.g., lipopolysaccharides), plants (e.g., xyloglucans) and animals (e.g., glycolipids, glycoproteins, and oligosaccharides). The biosynthetic pathway of GDP-L-fucose starts with a dehydration of GDP-D-mannose catalyzed by GDP-D-mannose 4,6-dehydratase (Gmd) creating GDP-4-keto-6-deoxymannose which is subsequently converted by the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (WcaG; GDP-beta-L-fucose synthetase) to GDP-beta-L-fucose. Both biosynthetic genes gmd and wcaG were cloned from Escherichia coli K12 and the enzymes overexpressed under control of the T7 promoter in the expression vectors pET11a and pET16b, yielding both native and N-terminal His-tag fusion proteins, respectively. The activities of the Gmd and WcaG were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-beta-L-fucose was optimized and the final product was purified. The formation of GDP-beta-L-fucose by the recombinant enzymes was verified by HPLC and NMR analyses. The His-tag fusion variants of the Gmd and WcaG proteins were purified to near homogeneity. The His-tag Gmd recombinant enzyme was inactive, whereas His-tag WcaG showed very similar enzymatic properties relative to the native GDP-beta-L-fucose synthetase. With the purified His-tag WcaG Km and Vmax values, respectively, of 40 microM and 23 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose and of 21 microM and 10 nkat/mg protein for the cosubstrate NADPH were obtained; a pH optimum of 7.5 was determined and the enzyme was stimulated to equal extend by the divalent cations Mg2+ and Ca2+. The Gmd enzyme showed a strong feedback inhibition by GDP-beta-L-fucose.
...
PMID:Preparative synthesis of GDP-beta-L-fucose by recombinant enzymes from enterobacterial sources. 1098 49

Fucosylation of glycans on glycoproteins and -lipids requires the enzymatic activity of relevant fucosyltransferases and GDP-L-fucose as the donor. Due to the biological importance of fucosylated glycans, a readily accessible source of GDP-L-fucose would be required. Here we describe the construction of a stable recombinant S.cerevisiae strain expressing the E.coli genes gmd and wcaG encoding the two enzymes, GDP-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GMER(FX)) respectively, needed to convert GDP-mannose to GDP-fucose via the de novo pathway. Taking advantage of the rich inherent cytosolic GDP-mannose pool in S.cerevisiae cells we could easily produce 0.2 mg/l of GDP-L-fucose with this recombinant yeast strain without addition of any external GDP-mannose. The GDP-L-fucose product could be used as the fucose donor for alpha1,3fucosyltransferase to synthesize sialyl Lewis x (sLex), a glycan crucial for the selectin-dependent leukocyte traffic.
...
PMID:Functional expression of Escherichia coli enzymes synthesizing GDP-L-fucose from inherent GDP-D-mannose in Saccharomyces cerevisiae. 1103 Jul 50

The enzymatic synthesis of GDP-beta-L-fucose and its enzymatic transfer reaction using recombinant enzymes from bacterial sources was examined. The GDP-D-mannose 4,6-dehydratase and the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase from Escherichia coli K-12, respectively, were used to catalyse the conversion of GDP-alpha-D-mannose to GDP-beta-L-fucose with 78% yield. For the transfer of the L-fucose to an acceptor, we cloned and overproduced the alpha-(1-->2)-fucosyltransferase (FucT2) protein from Helicobacter pylori. We were able to synthesise 2'-fucosyllactose using the overproduced FucT2 enzyme, enzymatically synthesised GDP-L-fucose and lactose. The isolation of 2'-fucosyllactose was accomplished by anion-exchange chromatography and gel filtration to give 65% yield.
...
PMID:Synthesis of the milk oligosaccharide 2'-fucosyllactose using recombinant bacterial enzymes. 1150 65

The whole genome approach enables the characterization of all components of any given biological pathway. Moreover, it can help to uncover all the metabolic routes for any molecule. Here we have used the genome of Drosophila melanogaster to search for enzymes involved in the metabolism of fucosylated glycans. Our results suggest that in the fruit fly GDP-fucose, the donor for fucosyltransferase reactions, is formed exclusively via the de novo pathway from GDP-mannose through enzymatic reactions catalyzed by GDP-D-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (GMER, also known as FX in man). The Drosophila genome does not have orthologs for the salvage pathway enzymes, i.e. fucokinase and GDP-fucose pyrophosphorylase synthesizing GDP-fucose from fucose. In addition we identified two novel fucosyltransferases predicted to catalyze alpha1,3- and alpha1,6-specific linkages to the GlcNAc residues on glycans. No genes with the capacity to encode alpha1,2-specific fucosyltransferases were found. We also identified two novel genes coding for O-fucosyltransferases and a gene responsible for a fucosidase enzyme in the Drosophila genome. Finally, using the Drosophila CG4435 gene, we identified two novel human genes putatively coding for fucosyltransferases. This work can serve as a basis for further whole-genome approaches in mapping all possible glycosylation pathways and as a basic analysis leading to subsequent experimental studies to verify the predictions made in this work.
...
PMID:Composition of Drosophila melanogaster proteome involved in fucosylated glycan metabolism. 1169 3

At least three structural proteins in Paramecium bursaria Chlorella virus (PBCV-1) are glycosylated, including the major capsid protein Vp54. However, unlike other glycoprotein-containing viruses that use host-encoded enzymes in the endoplasmic reticulum-Golgi to glycosylate their proteins, PBCV-1 encodes at least many, if not all, of the glycosyltransferases used to glycosylate its structural proteins. As described here, PBCV-1 also encodes two open reading frames that resemble bacterial and mammalian enzymes involved in de novo GDP-L-fucose biosynthesis. This pathway, starting from GDP-D-mannose, consists of two sequential steps catalyzed by GDP-D-mannose 4,6 dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, respectively. The two PBCV-1-encoded genes were expressed in Escherichia coli, and the recombinant proteins had the predicted enzyme activity. However, in addition to the dehydratase activity, PBCV-1 GMD also had a reductase activity, producing GDP-D-rhamnose. In vivo studies established that PBCV-1 GMD and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase are expressed after virus infection and that both GDP-L-fucose and GDP-D-rhamnose are produced in virus-infected cells. Thus, PBCV-1 is the first virus known to encode enzymes involved in nucleotide sugar metabolism. Because fucose and rhamnose are components of the glycans attached to Vp54, the pathway could circumvent a limited supply of GDP sugars by the algal host.
...
PMID:Paramecium bursaria Chlorella virus 1 encodes two enzymes involved in the biosynthesis of GDP-L-fucose and GDP-D-rhamnose. 1267 42

We cloned the GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase gene from Arabidopsis thaliana (AtFX/GER1). The yeast Saccharomyces cerevisiae was transfected with the AtFX/GER1 gene coexpressed with GDP-mannose-4,6-dehydratase gene of A. thaliana (MUR1). In vitro GDP-fucose synthesis activity was observed in the cytoplasmic fraction of cells coexpressing the AtFX/GER1 gene and MUR1 gene. However, the cytoplasmic fraction of cells expressing MUR1 alone did not show the GDP-mannose-4,6-dehydratase activity. This result suggests that the AtFX/GER1 protein may contribute to maintenance of the MUR1 protein as the active form. Immunoprecipitation experiments showed that both proteins interact with each other, indicating that this interaction is required to maintain MUR1 protein as the active or stable form. Finally, in vivo GDP-fucose synthesis activity was analyzed by measuring the amount of GDP-fucose produced in the cytoplasm of yeast cells. The amount of GDP-fucose in cells coexpressing MUR1 and AtFX/GER1 genes was 3.5 times higher than the amount of GDP-mannose in the same cells, indicating that this coexpression system is suitable for production of the valuable sugar nucleotide GDP-fucose in yeast.
...
PMID:Interaction of GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase with GDP-mannose-4,6-dehydratase stabilizes the enzyme activity for formation of GDP-fucose from GDP-mannose. 1288 8

d-Rhamnose is a rare 6-deoxy monosaccharide primarily found in the lipopolysaccharide of pathogenic bacteria, where it is involved in host-bacterium interactions and the establishment of infection. The biosynthesis of d-rhamnose proceeds through the conversion of GDP-d-mannose by GDP-d-mannose 4,6-dehydratase (GMD) to GDP-4-keto-6-deoxymannose, which is subsequently reduced to GDP-d-rhamnose by a reductase. We have determined the crystal structure of GMD from Pseudomonas aeruginosa in complex with NADPH and GDP. GMD belongs to the NDP-sugar modifying subfamily of the short-chain dehydrogenase/reductase (SDR) enzymes, all of which exhibit bidomain structures and a conserved catalytic triad (Tyr-XXX-Lys and Ser/Thr). Although most members of this enzyme subfamily display homodimeric structures, this bacterial GMD forms a tetramer in the same fashion as the plant MUR1 from Arabidopsis thaliana. The cofactor binding sites are adjoined across the tetramer interface, which brings the adenosyl phosphate moieties of the adjacent NADPH molecules to within 7 A of each other. A short peptide segment (Arg35-Arg43) stretches into the neighboring monomer, making not only protein-protein interactions but also hydrogen bonding interactions with the neighboring cofactor. The interface hydrogen bonds made by the Arg35-Arg43 segment are generally conserved in GMD and MUR1, and the interacting residues are highly conserved among the sequences of bacterial and eukaryotic GMDs. Outside of the Arg35-Arg43 segment, residues involved in tetrameric contacts are also quite conserved across different species. These observations suggest that a tetramer is the preferred, and perhaps functionally relevant, oligomeric state for most bacterial and eukaryotic GMDs.
...
PMID:Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway. 1473 33


1 2 Next >>

---