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:2.4.99.7 (sialyltransferase)
1,534 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Smooth membrane preparations of 13-day embryonic chicken livers, characterized by electron microscopy and marker enzyme analyses, have been found to contain sialyltransferase activity which displayed precise acceptor specificity. One sialyltransferase transferred N-acetyl-neuraminic acid (NANA) and gal beta 1 leads to 4glycNAc beta 1 leads R structures. Evidence based on competition studies suggests that a second enzyme is present transferring this sugar to a gal beta 1 leads to 3gal-NAc alpha 1 leads to R structure. The enzyme capable of adding NANA to gal beta 1 leads to 4glcNAc beta 1 leads to R structures has a pH optimum of 5.5, a temperature optimum of 30 degrees C, and half-saturating values of 17 muM for CMP-NANA and 180 muM for galactoside termini on desialyzed alpha 1-acid glycoprotein. It is activated about 10-fold by Triton X-100, has no exogenous divalent cation requirement, and is inhibited by CTP, CDP, and CMP. The enzyme requires carbohydrate structures underlying the gal beta 1 leads to 4glcNAc terminus for maximal catalytic activity; the necessity of such precise specificities of sialyltransferases is discussed in the light of recent structural evidence for the carbohydrate moieties of several glycoproteins.
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PMID:The specificity of sialyltransferase activity in smooth membrane fractions of embryonic chicken liver. 722 24

CMP-NAcNeu:GM3 ganglioside sialyltransferase (GD3 synthase) was characterized with respect to regulation of activity by nucleotides and compared in this regard with other sialyltransferases of ganglioside biosynthesis. Nucleotides preferentially inhibited the activity of GD3 synthase. Di- and trinucleotides inhibited most strongly and cytidine nucleotides were the most inhibitory class. The mode of inhibition by CMP (competitive or noncompetitive) varied with storage conditions of Golgi apparatus membranes; CMP inhibition was decreased during a series of consecutive freeze-thawings of membranes. Also, GD3 synthase inhibition by CDP was only partially relieved by excess Mg2+. With lactosylceramide as the in vitro precursor, synthesis of GM3 was always less inhibited by cytidine nucleotides than was that of GD3 and GT3. An 8-fold reduction in the ratio GD3/GM3 in the reaction products was obtained at 1.5 mM CTP. Separate incubations for the sialylation of GM3 or GM1 showed cytidine nucleotides increased synthesis of GD1a relative to GD3 by 3.5-fold.
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PMID:Ganglioside biosynthesis in rat liver: alteration of sialyltransferase activities by nucleotides. 740 17

All members of the sialyltransferase gene family cloned to date contain a conserved region, the "sialylmotif," consisting of 48-49 amino acids in the center of the coding sequence. To investigate the function of this motif, mutant constructs of the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase were designed by site-directed mutagenesis, replacing 11 individual conserved amino acids with alanine. Each of the mutants was expressed in COS-1 cells, and eight of these retained sialyltransferase activity, allowing comparison of their enzymatic properties with that of the wild type enzyme. Kinetic analysis showed that six of eight mutants had a 3-12-fold higher Km for the donor substrate CMP-NeuAc relative to the wild type enzyme, while the Km values for the acceptor substrate were within 0.5-1.2-fold of the wild type for all eight mutants evaluated. The Ki of the donor substrate analog CDP was also evaluated for the recombinant sialyltransferase with the Val to Ala mutation at residue 220, which produced a 6-fold increase in Km of CMP-NeuAc. A corresponding increase in Ki of 3.4-fold was observed for CDP, indicating a decreased affinity for the cytidine nucleotide. Taken together, these results suggest that the conserved sialylmotif in the sialyltransferase gene family participates in the binding of the common donor substrate, CMP-NeuAc.
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PMID:The sialyltransferase "sialylmotif" participates in binding the donor substrate CMP-NeuAc. 782 76

CMP-N-acetylneuraminic acid:lactosylceramide (alpha 2-3) sialyltransferase (GM3-synthase) was purified to homogeneity from a Triton CF-54 extract of young rat brain. The enzyme was separated by affinity chromatography on CDP-Sepharose column and resolved by linear NaCl gradient elution from the same adsorbent. Final purification of GM3-synthase was achieved by chromatography on a "lactosylceramide acid"-Sepharose column and specific elution with lactosylceramide. The enzyme activity was highest at pH 6.5 and required the presence of Triton CF-54 (0.15%) and Mn2+ (10 mM) for its full activity. The product of the reaction catalyzed by the enzyme was identified as GM3 based on its mobility on thin layer chromatographic plates using two different solvent systems. Comparison with several glycolipid substrates showed high specificity of GM3-synthase for lactosylceramide. The apparent Km value for lactosylceramide and CMP-N-acetylneuraminic acid were 80 and 210 microM, respectively. The apparent molecular mass of the enzyme determined on SDS-polyacrylamide gel electrophoresis was 76 kDa.
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PMID:Purification and characterization of CMP-N-acetylneuraminic acid:lactosylceramide (alpha 2-3) sialyltransferase (GM3-synthase) from rat brain. 825 49

A sensitive assay for sialyltransferase (STase activity extracted from gonococci with 0.5% Triton X100 was developed. Enzyme activity was optimal in the pH range 5.8-8.0 and was strongly inhibited by CMP, CDP and CTP, but not by other nucleotides, 10 mM Mg2+, Zn2+, Ca2+ or Mn2+, or by 18 mM EDTA. More than 90% of the activity was lost after 30 s at 67 degrees C. The apparent Vmax and apparent Km of the STase for cytidine 5'-monophospho-N-acetylneuraminic acid were 1.7 nmol of NANA incorporated/min/mg protein and 5.3 microM, respectively.
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PMID:Detection and some properties of the sialyltransferase implicated in the sialylation of lipopolysaccharide of Neisseria gonorrhoeae. 832 54

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

ST8Sia II (STX) and ST8Sia IV (PST) are polysialic acid (polySia) synthases that catalyze polySia formation of neural cell adhesion molecule (NCAM) in vivo and in vitro. It still remains unclear how these structurally similar enzymes act differently in vivo. In the present study, we performed the enzymatic characterization of ST8Sia II and IV; both ST8Sia II and IV have pH optima of 5.8-6.1 and have no requirement of metal ions. Because the pH dependence of ST8Sia II and IV enzyme activities and the pK profile of His residues are similar, we hypothesized that a histidine residue would be involved in their catalytic activity. There is a conserved His residue (cf. His(348) in ST8Sia II and His(331) in ST8Sia IV, respectively) within the sialyl motif VS in all sialyltransferase genes cloned to date. Mutant ST8Sia II and IV enzymes in which this His residue was changed to Lys showed no detectable enzyme activity, even though they were folded correctly and could bind to CDP-hexanolamine, suggesting the importance of the His residue for their catalytic activity. Next, the degrees of polymerization of polySia in NCAM catalyzed by ST8Sia II and IV were compared. ST8Sia IV catalyzed larger polySia formation of NCAM than ST8Sia II. We also analyzed the (auto)polysialylated enzymes themselves. Interestingly, when ST8Sia II or IV itself was sialylated under conditions for polysialylation, the disialylated compound was the major product, even though polysialylated compounds were also observed. These results suggested that both ST8Sia II and IV catalyze polySia synthesis toward preferred acceptor substrates such as NCAM, whereas they mainly catalyze disialylation, similarly to ST8Sia III, toward unfavorable substrates such as enzyme themselves.
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PMID:Differential biosynthesis of polysialic or disialic acid Structure by ST8Sia II and ST8Sia IV. 1127 64

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

Sialyltransferases transfer sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to an acceptor molecule. Trans-sialidases of parasites transfer alpha2,3-linked sialic acid from one molecule to another without the involvement of CMP-NeuAc. Here we report another type of sialylation, termed reverse sialylation, catalyzed by mammalian sialyltransferase ST3Gal-II. This enzyme synthesizes CMP-NeuAc by transferring NeuAc from the NeuAcalpha2,3Galbeta1,3GalNAcalpha unit of O-glycans, 3-sialyl globo unit of glycolipids, and sialylated macromolecules to 5'-CMP. CMP-NeuAc produced in situ is utilized by the same enzyme to sialylate other O-glycans and by other sialyltransferases such as ST6Gal-I and ST6GalNAc-I, forming alpha2,6-sialylated compounds. ST3Gal-II also catalyzed the conversion of 5'-uridine monophosphate (UMP) to UMP-NeuAc, which was found to be an inactive sialyl donor. Reverse sialylation proceeded without the need for free sialic acid, divalent metal ions, or energy. Direct sialylation with CMP-NeuAc as well as the formation of CMP-NeuAc from 5'-CMP had a wide optimum range (pH 5.2-7.2 and 4.8-6.4, respectively), whereas the entire reaction comprising in situ production of CMP-NeuAc and sialylation of acceptor had a sharp optimum at pH 5.6 (activity level 50% at pH 5.2 and 6.8, 25% at pH 4.8 and 7.2). Several properties distinguish forward/conventional versus reverse sialylation: (i) sodium citrate inhibited forward sialylation but not reverse sialylation; (ii) 5'-CDP, a potent forward sialyltransferase inhibitor, did not inhibit the conversion of 5'-CMP to CMP-NeuAc; and (iii) the mucin core 2 compound 3-O-sulfoGalbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-benzyl, an efficient acceptor for ST3Gal-II, inhibited the conversion of 5'-CMP to CMP-NeuAc. A significant level of reverse sialylation activity is noted in human prostate cancer cell lines LNCaP and PC3. Overall, the study demonstrates that the sialyltransferase reaction is readily reversible in the case of ST3Gal-II and can be exploited for the enzymatic synthesis of diverse sialyl products.
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PMID:Reversible sialylation: synthesis of cytidine 5'-monophospho-N-acetylneuraminic acid from cytidine 5'-monophosphate with alpha2,3-sialyl O-glycan-, glycolipid-, and macromolecule-based donors yields diverse sialylated products. 1806 23

Sialyloligosaccharides are synthesised by various glycosyltransferases and sugar nucleotides. All of these nucleotides are diphosphate compounds except for cytidine-5'-monophosphosialic acid (CMP-Neu5Ac). To obtain an insight into why cytidine-5'-diphosphosialic acid (CDP-Neu5Ac) has not been used for the sialyltransferase reaction and why it is not found in biological organisms, the compound was synthesised. This synthesis provided the interesting finding that the carboxylic acid moiety of the sialic acid attacks the attached phosphate group. This interaction yields an activated anhydride between carboxylic acid and the phosphate group and leads to hydrolysis of the pyrophosphate linkage. The mechanism was demonstrated by stable isotope-labelling experiments. This finding suggested that CMP-Neu5Ac might also form the corresponding anhydride structure between carboxylic acid and phosphate, and this seems to be the reason why CMP-Neu5Ac is acid labile in relation to other sugar nucleotides. To confirm the role of the carboxylic acid, CMP-Neu5Ac derivatives in which the carboxylic acid moiety in the sialic acid was substituted with amide or ester groups were synthesised. These analogues clearly exhibited resistance to acid hydrolysis. This result indicated that the carboxylic acid of Neu5Ac is associated with its stability in solution. This finding also enabled the development of a novel chemical synthetic method for CMP-Neu5Ac and CMP-sialic acid derivatives.
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PMID:Unique self-anhydride formation in the degradation of cytidine-5'-monophosphosialic acid (CMP-Neu5Ac) and cytidine-5'-diphosphosialic acid (CDP-Neu5Ac) and its application in CMP-sialic acid analogue synthesis. 2159 21


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