Purification to Apparent Homogeneity and Properties of Pig Kidneyl-Fucose Kinase

Park, Sang Ho; Pastuszak, Irena; Drake, Richard; Elbein, Alan D.

doi:10.1074/jbc.273.10.5685

Cited by 67 publications

(57 citation statements)

References 14 publications

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“…As generalized hypofucosylation is a biochemical hallmark of cells derived from LAD II patients (1,20), we determined whether fucose incorporation into macromolecules was affected in cells obtained from this patient. EBVtransformed lymphoblasts were grown in the presence of [ 3 H]-fucose, which can be converted to GDP-L- [ 3 H]-fucose by the salvage pathway (29,30). As shown in Figure 1, A and B, whereas the amount of radiolabeled fucose bound to acidprecipitable macromolecules was comparable in control, mother, and father, incorporation into macromolecules in patient cells was severely impaired (20% of that observed in the other samples).…”

Section: Resultsmentioning

confidence: 99%

Impairment of the Golgi GDP-l-Fucose Transport and Unresponsiveness to Fucose Replacement Therapy in LAD II Patients

et al. 2001

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Section: Resultsmentioning

confidence: 99%

Impairment of the Golgi GDP-l-Fucose Transport and Unresponsiveness to Fucose Replacement Therapy in LAD II Patients

et al. 2001

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“…Known C-1 phosphorylating enzymes are limited to mainly three types ( Fig. 2): the glycogen phosphorylases (16), which convert glycogen (9) into D-glucose-1-phosphate (10); fucokinases, which transfer a phosphate from ATP to the anomeric position of L-fucose (12) to provide ␤-L-fucose-1-phosphate (13) (17); and the galactokinases (GalK), which catalyze the formation of ␣-D-galactose-1-phosphate (Gal-1-P, 15) from D-galactose (14) and ATP. Previous studies have revealed that GalK from various sources have a limited substrate scope (18)(19)(20)(21), and in all C-1 kinases studied thus far, a strict adherence to either D-sugars (GalK and glycogen phosphorylases) or L-sugars (as in fucokinase) was observed.…”

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confidence: 99%

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Hoffmeister

Yang²,

Liu³

et al. 2003

Proceedings of the National Academy of Sciences

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Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates. Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides. Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase GalK. From this approach, one particular GalK mutant carrying a single amino acid exchange (Y371H) displayed a surprisingly substantial degree of kinase activity toward sugars as diverse as D-galacturonic acid, D-talose, L-altrose, and L-glucose, all of which failed as wild-type GalK substrates. Furthermore, this mutant provides enhanced turnover of the small pool of sugars converted by the wild-type enzyme. Comparison of this mutation to the recently solved structure of Lactococcus lactis GalK begins to provide a blueprint for further engineering of this vital class of enzyme. In addition, the rapid access to such promiscuous sugar C-1 kinases will significantly enhance accessibility to natural and unnatural sugar-1-phosphates and thereby impact both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.galactokinase ͉ glycorandomization ͉ in vitro evolution ͉ enzyme M any clinically important medicines are derived from glycosylated natural products, the D-or L-sugar substituents of which often dictate their overall biological activity. This paradigm is found throughout the anticancer and antiinfective arenas with representative clinical examples (Fig. 1a), including enediynes (calicheamicin, 1), polyketides (doxorubicin, 2; erythromycin, 3), indolocarbazoles (staurosporine, 4), nonribosomal peptides (vancomycin, 5), polyenes (nystatin, 6), coumarins (novobiocin, 7), and cardiac glycosides (digitoxin, 8) (1-7). Given the importance of the sugars attached to these and other biologically significant metabolites, extensive effort has been directed in recent years toward altering sugars as a means to enhance or alter natural product-based therapeutics by both in vivo and in vitro approaches (ref. 8 and references therein). Among these, in vitro glycorandomization (IVG) makes use of the inherent or engineered substrate promiscuity of nucleotidylyltransferases and glycosyltransferases to activate and attach chemically synthesized sugar precursors to various natural product scaffolds (6,7,(9)(10)(11)(12)(13)(14)(15), the advantage of which is the ability to efficiently incorporate highly functionalized ''unnatural'' sugar substitutions into the corresponding natural product scaffold (Fig. 1b). In a recent demonstration of IVG, Ͼ50 analogs of 5 were generated, some of which displayed enhanced and distinct antibacterial profiles from the parent natural product (13).As starting materials, sugar phosphates play a key role in the entire IVG process. Thus, the ability to rapidly construct sugar phosphate ...

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“…15. The divalent cations were also necessary for the GDP-L-Fuc pyrophosphorylase activity of rAtFKGP (Table 2).…”

Section: Identification Of Bifunctional L-fucokinase/gdp-l-fucosementioning

confidence: 99%

“…The specific activity of rAtFKGP for L-fucokinase was estimated at 0.17 unit/mg protein and that for GDP-L-Fuc pyrophosphorylase at 0.71 unit/mg protein (here the protein content was assessed based on the color intensity of rAtFKGP stained with Coomassie Brilliant Blue R-250 on SDS-PAGE). The specific activities of rAtFKGP apparently differ from those (1.27 and 0.36 units/mg protein, respectively) of pig L-fucokinase (15) and GDP-L-Fuc pyrophosphorylase (18). As has been observed for many nucleotide sugar pyrophosphorylases from other sources, rAtFKGP catalyzed both synthesis of GDP-L-Fuc from L-Fuc-1-P and GTP (GTP:L-fucose-1-phosphate guanylyltransferase action, the forward direction), and pyrophosphorolysis producing L-Fuc-1-P and GTP from GDP-L-Fuc and PP i (GDP-L-Fuc pyrophosphorylase action, the reverse).…”

Section: Identification Of Bifunctional L-fucokinase/gdp-l-fucosementioning

confidence: 99%

“…The enzymes participating in the salvage reactions for free L-Fuc have been well characterized in mammals, particularly in pigs. A pig kidney L-fucokinase (EC 2.7.1.52) specifically converts free L-Fuc to L-Fuc-1-P using ATP as the phosphate donor (15). Based on the amino acid sequence, the L-fucokinase is categorized into the GHMP (Galactokinase, Homoserine kinase, Mevalonate kinase, Phosphomevalonate kinase) family, which includes galactokinase (EC 2.7.1.6) and L-arabinokinase (EC 2.7.1.46) found in seed plants (16,17).…”

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confidence: 99%

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A Bifunctional Enzyme with L-Fucokinase and GDP-L-fucose Pyrophosphorylase Activities Salvages Free L-Fucose in Arabidopsis

Kotake

Hojo

Tajima

et al. 2008

Journal of Biological Chemistry

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Monomeric sugars generated during the metabolism of polysaccharides, glycoproteins, and glycolipids are imported to the cytoplasm and converted to respective nucleotide sugars via monosaccharide 1-phosphates, to be reutilized as activated sugars. Because L-fucose (L-Fuc) is activated mainly in the form of GDP derivatives in seed plants, the salvage reactions for L-Fuc are expected to be independent from those for Glc, Gal, L-arabinose, and glucuronic acid, which are activated as UDP-sugars. For this study we have identified, in the genomic data base of Arabidopsis, the gene ( The monosaccharide L-fucose (L-Fuc) is found as a constituent of cell wall polysaccharides and sugar moieties of glycoproteins and has important physiological functions in seed plants.L-Fucosyl residues, for example, occur as nonreducing terminal residues attached through ␣-(132)-linkages to penultimate sugar residues in xyloglucan and arabinogalactan protein (1, 2). The L-fucosylated trisaccharide side chains of xyloglucan modulate the interaction of xyloglucan with cellulose microfibrils, thereby affecting the mechanical properties of plant cell walls (3, 4), whereas some N-glycans of plant glycoproteins have L-fucosyl residues attached through ␣-(133)-linkages to the proximal GlcNAc residues (sugars mentioned in this paper belong to the D-series unless otherwise noted.), which are characteristic of plants but are not found in mammals and are responsible for the immunogenicity of plant glycoproteins in mammals (5). L-Fucosyl residues are also attached through ␣-(134)-linkages to L-rhamnosyl residues in pectic rhamnogalacturonan II (RG-II) 2 in seed plants (6). These L-fucosyl residues are transferred onto the glycoconjugates by actions of respective L-fucosyltransferases, which use GDP-L-Fuc as the L-fucosyl donor. Recent studies have identified a xyloglucan-specific L-fucosyltransferase, AtFUT1 (7), and the related L-fucosyltransferase genes (AtFUT2-10) have also been found in the genome of Arabidopsis (8).GDP-L-Fuc, the activated form of L-Fuc, is generated through both de novo and salvage pathways (9, 10). Recently, the importance of levels of GDP-L-Fuc for the architecture of L-Fuc-containing cell wall polysaccharides was demonstrated through the study of the L-Fuc-deficient mutant of Arabidopsis, mur1. The mur1 mutant has reduced L-Fuc content in RG-II because of a defect in GDP-Man 4,6-dehydratase (EC 4.2.1.47) that catalyzes the first step of conversion of GDP-Man to GDP-L-Fuc in the de novo pathway (11,12). The loss of L-fucosyl residues indispensable for the boron-mediated dimer formation of pectic RG-II reduces the growth of rosette leaves in the mutant. The dwarf phenotype of mur1, however, can be rescued by exogenously applied monomeric L-Fuc, possibly because a compensating supply of GDP-L-Fuc is generated from L-Fuc via L-Fuc 1-phosphate (L-Fuc-1-P) in the salvage pathway (11). It is

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Purification to Apparent Homogeneity and Properties of Pig Kidneyl-Fucose Kinase

Cited by 67 publications

References 14 publications

Impairment of the Golgi GDP-l-Fucose Transport and Unresponsiveness to Fucose Replacement Therapy in LAD II Patients

Impairment of the Golgi GDP-l-Fucose Transport and Unresponsiveness to Fucose Replacement Therapy in LAD II Patients

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

A Bifunctional Enzyme with L-Fucokinase and GDP-L-fucose Pyrophosphorylase Activities Salvages Free L-Fucose in Arabidopsis

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