UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, functionally expressed in and purified from Escherichia coli, yeast, and insect cells

Blume, Astrid; Ghaderi, Darius; Liebich, Viola; Hinderlich, Stephan; Donner, Peter; Reutter, Werner; Lucka, Lothar

doi:10.1016/j.pep.2004.02.013

Cited by 16 publications

(14 citation statements)

References 33 publications

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“…The sizes of the N-terminal mutant proteins were 79 kDa, 58 kDa and 45 kDa, whereas the sizes of the C-terminal mutant proteins were 85 kDa, 83 kDa, 72 kDa, 61 kDa and 49 kDa. In insect cells, soluble, wild-type UDP-GlcNAc 2-epimerase/ManNAc kinase typically represented 30-50 % of the total protein expression [27]. Similar expression levels were observed for the 717-722 mutant.…”

Section: Generation Of Deletion Mutantsmentioning

confidence: 63%

“…In parallel, the amount of active UDP-GlcNAc 2-epimerase/ManNAc kinase in each fraction was determined by Western blotting. The specific activities of the aggregates were much lower than the activities of the respective native enzyme, as already shown earlier for the wild-type enzyme [27]. Therefore relative specific enzyme activities of the mutants with respect to the wild-type enzyme were determined only for fractions with proteins < 600 kDa ( Figure 3).…”

Section: Enzymic Activities Of the Deletion Mutantsmentioning

confidence: 73%

“…Rat cDNA of UDP-GlcNAc 2-epimerase/ManNAc kinase, cloned into the pFastBacHTA vector [27], was used as template DNA. The primers used are listed in Table 1.…”

Section: Generation Of Deletion Mutants Of Udp-glcnac 2-epimerase/manmentioning

confidence: 99%

“…Both the wild-type and the deletion mutants of UDP-GlcNAc 2-epimerase/ManNAc kinase were expressed in insect cells using the Bac-to-Bac ® System (GibcoBRL) as described previously [27]. In brief, constructs of the deletion mutants were transformed into DH10Bac E. coli cells for transposition into bacmid DNA.…”

Section: Expression Of Proteins In Sf9 Insect Cellsmentioning

confidence: 99%

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Domain-specific characteristics of the bifunctional key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

Blume

Weidemann

Stelzl

et al. 2004

Biochemical Journal

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UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is a bifunctional enzyme, which initiates and regulates sialic acid biosynthesis. Sialic acids are important compounds of mammalian glycoconjugates, mediating several biological processes, such as cell-cell or cell-matrix interactions. In order to characterize the function of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, a number of deletion mutants were generated, lacking either parts of the N-terminal epimerase or the C-terminal kinase domain. N-terminal deletion of only 39 amino acids results in a complete loss of epimerase activity. Deletions in the C-terminal part result in a reduction or complete loss of kinase activity, depending on the size of the deletion. Deletions at either the N-or the C-terminus also result in a reduction of the other enzyme activity. These results indicate that a separate expression of both domains is possible, but that a strong intramolecular dependency of the two domains has arisen during evolution of the enzyme. N-terminal, as well as C-terminal, mutants tend to form trimers, in addition to the hexameric structure of the native enzyme. These results and yeast two-hybrid experiments show that structures required for dimerization are localized within the kinase domain, and a potential trimerization site is possibly located in a region between the two domains.In conclusion, our results reveal that the activities, as well as the oligomeric structure, of this bifunctional enzyme seem to be organized and regulated in a complex manner.

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Section: Generation Of Deletion Mutantsmentioning

confidence: 63%

Section: Enzymic Activities Of the Deletion Mutantsmentioning

confidence: 73%

“…Rat cDNA of UDP-GlcNAc 2-epimerase/ManNAc kinase, cloned into the pFastBacHTA vector [27], was used as template DNA. The primers used are listed in Table 1.…”

Section: Generation Of Deletion Mutants Of Udp-glcnac 2-epimerase/manmentioning

confidence: 99%

Section: Expression Of Proteins In Sf9 Insect Cellsmentioning

confidence: 99%

See 2 more Smart Citations

Domain-specific characteristics of the bifunctional key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

Blume

Weidemann

Stelzl

et al. 2004

Biochemical Journal

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“…Expression and Purification of UDP-GlcNAc 2-Epimerase/ManNAc Kinase-Rat UDP-GlcNAc 2-epimerase/ManNAc kinase was recombinantly expressed in Sf-900 insect cells with a His 6 tag as previously described (31). Cells from a 50-ml culture were harvested, resuspended in 5 ml of 10 mM sodium phosphate, 1 mM EDTA, 1 mM dithiothreitol, pH 7.5, and lysed by passage through a narrow bore needle 20 times.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Ligand Binding to the Bifunctional Key Enzyme in the Sialic Acid Biosynthesis by NMR

Blume

Benie

Stolz

et al. 2004

Journal of Biological Chemistry

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The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme for the biosynthesis of sialic acids. As terminal components of glycoconjugates, sialic acids are associated with a variety of pathological processes such as inflammation and cancer. For the first time, this study reveals characteristics of the interaction of the epimerase site of the enzyme with its natural substrate, UDP-N-acetylglucosamine (UDP-GlcNAc) and derivatives thereof at atomic resolution. Saturation transfer difference NMR experiments were crucial in obtaining ligand binding epitopes and to rank ligands according to their binding affinities. Employing a fragment based approach, it was possible to assign the major component of substrate recognition to the UDP moiety. In particular, the binding epitopes of the uridine moieties of UMP, UDP, UDP-GalNAc, and UDP-GlcNAc are rather similar, suggesting that the binding mode of the UDP moiety is the same in all cases. In contrast, the hexopyranose units of UDP-GlcNAc and UDP-GalNAc display small differences reflecting the inability of the enzyme to process UDP-GalNAc. Surprisingly, saturation transfer difference NMR titrations show that UDP has the largest binding affinity to the epimerase site and that at least one phosphate group is required for binding. Consequently, this study provides important new data for rational drug design.

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UDP-GlcNAc 2-Epimerase/ManNAc Kinase (GNE)

Reutter

Hinderlich

Kemmner

2014

Handbook of Glycosyltransferases and Related Genes

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UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, functionally expressed in and purified from Escherichia coli, yeast, and insect cells

Cited by 16 publications

References 33 publications

Domain-specific characteristics of the bifunctional key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

Domain-specific characteristics of the bifunctional key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

Characterization of Ligand Binding to the Bifunctional Key Enzyme in the Sialic Acid Biosynthesis by NMR

UDP-GlcNAc 2-Epimerase/ManNAc Kinase (GNE)

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