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

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Query: EC:2.4.99.6 (sialyltransferase)
1,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the potential of filamentous fungi to synthesize N-glycans that are convertible to a mammalian type, in vitro glycosylation assays were performed. Recombinant human N-acetylglucosaminyltransferase I, human beta-1,4-galactosyltransferase and rat alpha-2,6-sialyltransferase were successively used to mimic part of the mammalian glycosylation synthesis pathway. High-mannose carbohydrates on Trichoderma reesei cellobiohydrolase I were converted to a hybrid mammalian-type structure. Successful modification varied markedly with the strain of T. reesei used to produce cellobiohydrolase I. In vitro pretreatment of fungal glycoproteins with Aspergillus saitoi alpha-1,2-mannosidase improved subsequent hybrid formation. It was, however, not possible to trim all fungal oligosaccharides to an acceptor substrate for mammalian glycosyltransferases. With T. reesei RUTC 30, capping glucose residues and phosphate groups were shown to be responsible for this lack of trimming. N-glycan processing in T. reesei apparently involves different steps, including alpha-1,2-mannosidase trimmings, and thus resembles the first mammalian glycosylation processes. The alpha-1,2-mannosidase trimming steps can be exploited for further in vitro and/or in vivo synthesis of complex oligosaccharides on (heterologous) glycoproteins from filamentous fungi.
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PMID:In vitro conversion of the carbohydrate moiety of fungal glycoproteins to mammalian-type oligosaccharides--evidence for N-acetylglucosaminyltransferase-I-accepting glycans from Trichoderma reesei. 939 16

Quinic acid (4) was transformed into phosphitamides 6, 14, and 15, which could be readily linked to 5'-O-unprotected cytidine derivative 7; ensuing oxidation of the obtained phosphite triesters with tert-butylhydroperoxide furnished the corresponding phosphate triesters 8, 16, and 17, respectively. Hydrogenolytic debenzylation of the phosphate moiety, base catalysed removal of acetyl protective groups, and basic hydrolysis of the methylester of the quinic acid moiety furnished CMP-Neu5Ac analogues 1-3. In order to measure their inhibition of sialyltransferases, a nonradioactive sialyltransferase assay [employed for alpha(2-6)-sialyltransferase from rat liver (EC 2.4.99.1)] based on reversed-phase HPLC separation of UV-labelled acceptor 20 (p-nitrophenyl glycoside of N-acetyllactosamine) from the UV-labelled product 21 (p-nitrophenyl glycoside of sialyl alpha(2-6')-N-acetyllactosamine) and p-nitrophenylalanine as internal standard was developed. The assay reproduced the reported K(M) values for CMP-Neu5Ac and N-acetyllactosamine and the Ki values for CDP. 1 and 2 turned out to be potent sialyltransferase inhibitors.
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PMID:New sialyltransferase inhibitors based on CMP-quinic acid: development of a new sialyltransferase assay. 961 21

Sialic acids from the liver and serum of guinea-pig are composed of N-acetylneuraminic acid (Neu5Ac; 85% and 61%, respectively), N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2; 10% and 32%, respectively) and N-glycolylneuraminic acid (Neu5Gc; 5% and 7%, respectively), besides traces of N-glycolyl-4-O-acetylneuraminic acid in serum. The analysis was carried out using thin-layer chromatography, high-performance liquid chromatography, electron impact ionization mass spectrometry, and different enzymes (sialidase, sialate esterase, and sialate-pyruvate lyase after hydrolysis and purification of the sialic acids by ion-exchange chromatography). We showed that this O-acetylation of sialic acids is due to the activity of an acetyl-coenzyme A:sialate-4-O-acetyltransferase (EC 2.3.1.44), which occurs together with sialyltransferase activity in Golgi-enriched membrane fractions of guinea-pig liver. The enzyme operates optimally at 30 degrees C in 70 mM potassium phosphate buffer at pH 6.7 and in the presence of 90 mM KCI with an apparent KM for AcCoA of 0.6 1microM and a Vmax of 20 pmol/mg protein x min. The enzyme is inhibited by coenzyme A in a mixed-competitive manner (Ki = 4.2 microM), as well as by parachloromercuribenzoate, MnCl2, saponin and Triton X-100.
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PMID:Enzymatic 4-O-acetylation of N-acetylneuraminic acid in guinea-pig liver. 1005 93

D-Glucosamine was transformed into phenyl and 2-benzoyloxyethyl N-acetylglucosamine beta-glycosides 6a and 6b, respectively. Transformation of 6a,b into 6-O-unprotected N-acetylglucosamine derivatives 9a,b permitted the generation of an aldehyde group in the 6-position. Treatment of these intermediates with base afforded unsaturated aldehyde derivatives 10a,b, which are structural mimics of 2,3-dehydroneuraminic acid. H-Phosphonate addition to the aldehyde group and attachment of the cytidine monophosphate residue to the generated hydroxy group gave fully protected transition state analogues of cytidine monophosphate-N-acetylneuraminic acid 14a,b. Liberation of the unprotected compounds 1ah,l and 1bh,l led to excellent inhibitors of alpha(2-6)-sialyltransferase from rat liver. Variation of the protective group cleavage procedure for 14a,b led to formal loss of phosphate, thus resulting in diene derivatives (E)-/(Z)-2a,b, which also exhibited inhibitory properties.
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PMID:Efficient sialyltransferase inhibitors based on glycosides of N-acetylglucosamine. 1185 37

An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5'-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of alpha2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and alpha2,3-sialyltransferase.
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PMID:Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst. 1235 62

Hepatitis B virus MHBst and HBx fragments were amplified to construct eukaryotic expression vector pCDNA3.1-MHBst and pCDNA3.1-HBx. ST3GalI promoter region was obtained by the method of PCR and GFP report plasmid pEGFP-N1-Psial was constructed. pCDNA3.1-MHBst or pCDNA3.1-HBx with pEGFP-N1-Psial were transiently co-transfected into QGY-7701 cells using calcium phosphate-DNA co-precipitation, respectively. The expressions of Psial-directed GFP were analyzed by FAC-Scalibur. It was found that MHBst/HBx could upregulate ST3GalI promoter activity by 35.2% and 43.8%, respectively. We report the regulation of ST3GalI by MHBst and HBx transactivators. It would be helpful to further investigate the relation between hepatitis B virus infection and sialyltransferase expression.
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PMID:[Regulation of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase (ST3GalI) by hepatitis B virus MHBst/HBx transactivator]. 1256 Nov 97

Sialic acid terminates oligosaccharide chains on mammalian and microbial cell surfaces, playing critical roles in recognition and adherence. The enzymes that transfer the sialic acid moiety from cytidine-5'-monophospho-N-acetyl-neuraminic acid (CMP-NeuAc) to the terminal positions of these key glycoconjugates are known as sialyltransferases. Despite their important biological roles, little is understood about the mechanism or molecular structure of these membrane-associated enzymes. We report the first structure of a sialyltransferase, that of CstII from Campylobacter jejuni, a highly prevalent foodborne pathogen. Our structural, mutagenesis and kinetic data provide support for a novel mode of substrate binding and glycosyl transfer mechanism, including essential roles of a histidine (general base) and two tyrosine residues (coordination of the phosphate leaving group). This work provides a framework for understanding the activity of several sialyltransferases, from bacterial to human, and for the structure-based design of specific inhibitors.
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PMID:Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog. 1473 Mar 52

Sialyltransferases catalyze reactions that transfer a sialic acid from CMP-sialic acid to an acceptor (a structure terminated with galactose, N-acetylgalactosamine, or sialic acid). They are key enzymes that catalyze the synthesis of sialic acid-containing oligosaccharides, polysaccharides, and glycoconjugates that play pivotal roles in many critical physiological and pathological processes. The structures of a truncated multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1), in the absence and presence of CMP, have been determined by X-ray crystallography at 1.65 and 2.0 A resolutions, respectively. The Delta24PmST1 exists as a monomer in solution and in crystals. Different from the reported crystal structure of a bifunctional sialyltransferase CstII that has only one Rossmann domain, the overall structure of the Delta24PmST1 consists of two separate Rossmann nucleotide-binding domains. The Delta24PmST1 structure, thus, represents the first sialyltransferase structure that belongs to the glycosyltransferase-B (GT-B) structural group. Unlike all other known GT-B structures, however, there is no C-terminal extension that interacts with the N-terminal domain in the Delta24PmST1 structure. The CMP binding site is located in the deep cleft between the two Rossmann domains. Nevertheless, the CMP only forms interactions with residues in the C-terminal domain. The binding of CMP to the protein causes a large closure movement of the N-terminal Rossmann domain toward the C-terminal nucleotide-binding domain. Ser 143 of the N-terminal domain moves up to hydrogen-bond to Tyr 388 of the C-terminal domain. Both Ser 143 and Tyr 388 form hydrogen bonds to a water molecule, which in turn hydrogen-bonds to the terminal phosphate oxygen of CMP. These interactions may trigger the closure between the two domains. Additionally, a short helix near the active site seen in the apo structure becomes disordered upon binding to CMP. This helix may swing down upon binding to donor CMP-sialic acid to form the binding pocket for an acceptor.
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PMID:Cytidine 5'-monophosphate (CMP)-induced structural changes in a multifunctional sialyltransferase from Pasteurella multocida. 1647 3

Sialyltransferases are a family of glycosyltransferases that catalyze the transfer of N-acetylneuraminic acid residues from cytidine monophosphate N-acetylneuraminic acid (CMP-NeuAc) as a donor substrate to the carbohydrate groups of glycoproteins and glycolipids as acceptor substrates. We determined the crystal structure of Delta16psp26ST, the N-terminal truncated form of alpha2,6-sialyltransferase from Vibrionaceae Photobacterium sp. JT-ISH-224, complexed with a donor product CMP and an acceptor substrate lactose. Delta16psp26ST has three structural domains. Domain 1 belongs to the immunoglobulin-like beta-sandwich fold, and domains 2 and 3 form the glycosyltransferase-B structure. The CMP and lactose were bound in the deep cleft between domains 2 and 3. In the structure, only Asp232 was within hydrogen-binding distance of the acceptor O6 carbon of the galactose residue in lactose, and His405 was within hydrogen-binding distance of the phosphate oxygen of CMP. Mutation of these residues greatly decreased the activity of the enzyme. These structural and mutational results indicated that Asp232 might act as a catalytic base for deprotonation of the acceptor substrate, and His405 might act as a catalytic acid for protonation of the donor substrate. These findings are consistent with an in-line-displacement reaction mechanism in which Delta16psp26ST catalyzes the inverting transfer reaction. Unlike the case with multifunctional sialyltransferase (Delta24PmST1) complexed with CMP and lactose, the crystal structure of which was recently reported, the alpha2,6 reaction specificity of Delta16psp26ST is likely to be determined by His123.
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PMID:Crystal structure of Vibrionaceae Photobacterium sp. JT-ISH-224 alpha2,6-sialyltransferase in a ternary complex with donor product CMP and acceptor substrate lactose: catalytic mechanism and substrate recognition. 1796 95

Sialylation of glycans is ubiquitous in vertebrates, but was believed to be absent in plants, arthropods, and fungi. However, recently evidence has been provided for the presence of sialic acid in these evolutionary clades. In addition, homologs of mammalian genes involved in sialylation can be found in the genomes of these taxa and for some Drosophila enzymes, involvement in sialic acid metabolism has been documented. In plant genomes, homologs of sialyltransferase genes have been identified, but there activity could not be confirmed. Several mammalian cell lines exist with defects in the sialylation pathway. One of these is the Chinese hamster ovary cell line Lec2, deficient in CMP-sialic acid transport to the Golgi lumen. These mutants provide the possibility to clone genes by functional complementation. Using expression cloning, we have identified an Arabidopsis thaliana nucleotide sugar transporter that is able to complement the CMP-sialic acid transport deficiency of Lec2 cells. The isolated gene (At5g41760) is a member of the triose-phosphate/nucleotide sugar transporter gene family. Recombinant expression of the gene in yeast and testing in vitro confirmed its ability to transport CMP-sialic acid.
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PMID:A CMP-sialic acid transporter cloned from Arabidopsis thaliana. 1825 24


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