共查询到20条相似文献,搜索用时 15 毫秒
1.
Elling L Zervosen A Gallego RG Nieder V Malissard M Berger EG Vliegenthart JF Kamerling JP 《Glycoconjugate journal》1999,16(7):327-336
The capacity of UDP-N-acetyl-alpha-D-glucosamine (UDP-GlcNAc) as an in vitro acceptor substrate for beta-1,4-galactosyltransferase (beta4GalT1, EC 2.4.1.38) from human and bovine milk and for recombinant human beta4GalT1, expressed in Saccharomyces cerevisiae, was evaluated. It turned out that each of the enzymes is capable to transfer Gal from UDP-alpha-D-galactose (UDP-Gal) to UDP-GlcNAc, affording Gal(beta1-4)GlcNAc(alpha1-UDP (UDP-LacNAc). Using beta4GalT1 from human milk, a preparative enzymatic synthesis of UDP-LacNAc was carried out, and the product was characterized by fast-atom bombardment mass spectrometry and 1H and 13C NMR spectroscopy. Studies with all three beta4GalTs in the presence of alpha-lactalbumin showed that the UDP-LacNAc synthesis is inhibited and that UDP-alpha-D-glucose is not an acceptor substrate. This is the first reported synthesis of a nucleotide-activated disaccharide, employing a Leloir glycosyltransferase with a nucleotide-activated monosaccharide as acceptor substrate. Interestingly, in these studies beta4GalT1 accepts an alpha-glycosidated GlcNAc derivative. The results imply that beta4GalT1 may be responsible for the biosynthesis of UDP-LacNAc, previously isolated from human milk. 相似文献
2.
Bovine beta-(1-->4)-galactosyltransferase was assayed with a series of 5a-carba-sugars, i.e., sugar analogues in which the ring oxygen of pyranose is replaced by a methylene group. The analogues are 5a-carba-sugar of 2-acetamido-2-deoxy-alpha-DL-galactopyranose, both alpha and beta anomers of 2-acetamido-2-deoxy-DL-glucopyranose (5a-carba-DL-GlcNAc), and 2-acetamido-2-deoxy-DL-mannopyranose. Of these analogues, both alpha and beta anomers of 5a-carba-DL-GlcNAc act as an acceptor. Enzymatic synthesis using the alpha and beta anomers of 5a-carba-DL-GlcNAc afforded the corresponding D-Gal-beta-(1-->4)-5a-carba-alpha-D-GlcNAc and D-Gal-beta-(1-->4)-5a-carba-beta-D-GlcNAc on a practical scale, and these structures were confirmed by NMR spectroscopy. These results indicate that the ring oxygen atom in the 5a-carba-D-GlcNAc is not used for specific recognition by bovine beta-(1-->4)-galactosyltransferase. 相似文献
3.
T Murata M Kosugi T Nakamura T Urashima T Usui 《Bioscience, biotechnology, and biochemistry》2001,65(11):2456-2464
We have established a unique enzymatic approach for obtaining sulfated disaccharides using Bacillus circulans beta-D-galactosidase-catalyzed 6-sulfo galactosylation. When 4-methyl umbelliferyl 6-sulfo beta-D-galactopyranoside (S6Gal beta-4MU) was used as a donor, the enzyme induced transfer of 6-sulfo galactosyl residue to GlcNAc acceptor. As a result, the desired compound 6'-sulfo N-acetyllactosamine (S6Gal beta1-4GlcNAc) and its positional isomer 6'-sulfo N-acetylisolactosamine (S6Gal beta1-6GlcNAc) were observed by HPAEC-PAD, in 49% total yield based on the donor added, and in a molar ratio of 1:3.5. With a glucose acceptor, the regioselectivity was substantially changed and S6Gal beta1-2Glc was mainly produced along with beta-(1-1)alpha, beta-(1-3), beta-(1-6) isomers in 74% total yield. When methyl alpha-D-glucopyranoside (Glc alpha-OMe) was an acceptor, the enzyme also formed mainly S6Gal beta1-2Glc alpha-OMe with its beta-(1-6)-linked isomer in 41% total yield based on the donor added. In both cases, it led to the predominant formation of beta-(1-2)-linked disaccharides. In contrast, with the corresponding methyl beta-D-glucopyranoside (Glc beta-OMe) acceptor, S6Gal beta1-3Glc beta-OMe and S6Gal beta1-6Glc beta-OMe were formed in a low total yield of 12%. These results indicate that the regioselectivity and efficiency on the beta-D-galactosidase-mediated transfer reaction significantly depend on the anomeric configuration in the glucosyl acceptors. 相似文献
4.
Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall 总被引:5,自引:0,他引:5
Fontaine T Simenel C Dubreucq G Adam O Delepierre M Lemoine J Vorgias CE Diaquin M Latgé JP 《The Journal of biological chemistry》2000,275(36):27594-27607
Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan. 相似文献
5.
A beta-(1-->6)-linked D-galactofuranosyl hexasaccharide was synthesized efficiently in a block construction manner by the well-known Schmidt glycosylation method using 6-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-galactofuranosyl trichloroacetimidate (1) and allyl 2,3,5-tri-O-benzoyl-beta-D-galactofuranoside (3) as the key synthons. Coupling of 3 with 1 gave beta-(1-->6)-linked disaccharide 4. Subsequent selective deacetylation of 4 afforded the disaccharide acceptor 5, while deallylation of 4 followed by trichloroacetimidate formation produced the disaccharide donor 6. Condensation of 5 with 6 gave the tetrasaccharide 7, and subsequent deacetylation afforded the tetrasaccharide acceptor 8. Finally, coupling of 8 with 6 followed by deacylation yielded the target beta-(1-->6)-linked galactofuranose hexasaccharide 10. All of the reactions in the synthesis were carried out smoothly and in high yield. 相似文献
6.
Large-scale screening of genetic and chemical-genetic interactions was used to examine the assembly and regulation of beta-1,3-glucan in Saccharomyces cerevisiae. Using the set of deletion mutants in approximately 4600 nonessential genes, we scored synthetic interactions with genes encoding subunits of the beta-1,3-glucan synthase (FKS1, FKS2), the glucan synthesis regulator (SMI1/KNR4), and a beta-1,3-glucanosyltransferase (GAS1). In the resulting network, FKS1, FKS2, GAS1, and SMI1 are connected to 135 genes in 195 interactions, with 26 of these genes also interacting with CHS3 encoding chitin synthase III. A network core of 51 genes is multiply connected with 112 interactions. Thirty-two of these core genes are known to be involved in cell wall assembly and polarized growth, and 8 genes of unknown function are candidates for involvement in these processes. In parallel, we screened the yeast deletion mutant collection for altered sensitivity to the glucan synthase inhibitor, caspofungin. Deletions in 52 genes led to caspofungin hypersensitivity and those in 39 genes to resistance. Integration of the glucan interaction network with the caspofungin data indicates an overlapping set of genes involved in FKS2 regulation, compensatory chitin synthesis, protein mannosylation, and the PKC1-dependent cell integrity pathway. 相似文献
7.
Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4Glc) and isoglobotetraose (GalNAcbeta1-->3Galalpha1-->3Galbeta1-->4Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant beta-1,3-N-acetylgalactosaminyltransferase from Haemophilus influenzae strain Rd can transfer N-acetylgalactosamine to a wide range of acceptor substrates with a terminal galactose residue. The donor substrate UDP-GalNAc can be regenerated by a six-enzyme reaction cycle consisting of phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, phosphate acetyltransferase, pyruvate kinase, and inorganic pyrophosphatase from Escherichia coli, as well as UDP-N-acetylglucosamine C4 epimerase from Plesiomonas shigelloides. All these enzymes were overexpressed in E. coli with six-histidine tags and were purified by one-step nickel-nitrilotriacetic acid affinity chromatography. Multiple-enzyme synthesis of globotetraose or isoglobotetraose with the purified enzymes was achieved with relatively high yields. 相似文献
8.
α-1,6-甘露糖转移酶基因敲除的毕赤酵母菌株构建及其用于融合蛋白HSA/GM-CSF 表达的研究 总被引:1,自引:0,他引:1
酵母对蛋白的糖基化修饰过程不同于哺乳动物,其特点为产生高甘露糖型糖基且易发生过度糖基化。本研究通过两步基因重组敲除目标基因的方法成功敲除了毕赤酵母中的α-1,6-甘露糖转移酶(och1p)基因,获得了och1敲除的菌株。以此为基础,构建了高效表达人血清白蛋白与粒细胞-巨噬细胞集落刺激因子融合蛋白(HSA/GM-CSF)的工程酵母,与野生型毕赤酵母表达的过度糖基化HSA/GM-CSF不同,och1敲除菌表达的该融合蛋白糖基化程度明显降低,这为该融合蛋白的开发提供了重要基础。och1敲除菌株的构建不仅提供了一个对糖蛋白进行低糖基化修饰的毕赤酵母表达系统,而且为进一步的酵母糖基工程改造提供了基础。 相似文献
9.
Separation and characterization of two alpha 1,2-mannosyltransferase activities from Saccharomyces cerevisiae 总被引:5,自引:0,他引:5
Two GDP-mannose-dependent mannosyltransferase activities (designated M1MT-I and M2MT-I) from Triton X-100 extracts of Saccharomyces cerevisiae mnn1 microsomes were separated by concanavalin A lectin chromatography and partially purified. The two transferases were distinguished by differences in concanavalin A affinity and in carbohydrate acceptor specificity. Analyses of the reaction products indicate that both enzymes are alpha 1,2-mannosyltransferases. M1MT-I utilizes mannose or methyl-alpha-mannoside as acceptor while M2MT-I catalyzes the transfer of mannose from GDP-mannose to unsubstituted nonreducing alpha 1,6-linked mannose residues in the acceptor molecule. M2MT-I activity correlates with the presence of a single alpha 1,2-linked mannose residue at the nonreducing terminus of mnn2mnn9 and mnn2mnn10 outer chain oligosaccharides, and the enzyme may be involved in regulating outer chain elongation. 相似文献
10.
Interaction of soluble glucosyl- and mannosyl-transferase enzyme activities in the synthesis of a glucomannan 总被引:3,自引:1,他引:2
A neutral-detergent-solubilized-enzyme preparation derived from Phaseolus aureus hypocotyls contains two types of glycosyltransferase activity. One, mannosyltransferase enzyme activity, utilizes GDP-alpha-d-mannose as the sugar nucleotide substrate. The other, glucosyltransferase enzyme activity, utilizes GDP-alpha-d-glucose as the sugar nucleotide substrate. The soluble enzyme preparation catalyses the formation of what appears to be a homopolysaccharide when either sugar nucleotide is the only substrate present. A beta-(1-->4)-linked mannan is the only polymeric product when only GDP-alpha-d-mannose is added. A beta-(1-->4)-linked glucan is the only polymeric product when only GDP-alpha-d-glucose is added. In the presence of both sugar nucleotides, however, a beta-(1-->4)-linked glucomannan is formed. There are indications that endogenous sugar donors may be present in the enzyme preparation. There appear to be only two glycosyltransferases in the enzyme preparation, each catalysing the transfer of a different sugar to the same type of acceptor molecule. The glucosyltransferase requires the continual production of mannose-containing acceptor molecules for maintenance of enzyme activity, and is thereby dependent upon the activity of the mannosyltransferase. The mannosyltransferase, on the other hand, does not require the continual production of glucose-containing acceptors for maintenance of enzyme activity, but is severely inhibited by GDP-alpha-P-glucose. These properties promote the synthesis of beta-(1-->4)-linked glucomannan rather than beta-(1-->4)-linked glucan plus beta-(1-->4)-linked mannan when both sugar nucleotide substrates are present. 相似文献
11.
Mille C Bobrowicz P Trinel PA Li H Maes E Guerardel Y Fradin C Martínez-Esparza M Davidson RC Janbon G Poulain D Wildt S 《The Journal of biological chemistry》2008,283(15):9724-9736
Structural studies of cell wall components of the pathogenic yeast Candida albicans have demonstrated the presence of beta-1,2-linked oligomannosides in phosphopeptidomannan and phospholipomannan. During C. albicans infection, beta-1,2-oligomannosides play an important role in host/pathogen interactions by acting as adhesins and by interfering with the host immune response. Despite the importance of beta-1,2-oligomannosides, the genes responsible for their synthesis have not been identified. The main reason is that the reference species Saccharomyces cerevisiae does not synthesize beta-linked mannoses. On the other hand, the presence of beta-1,2-oligomannosides has been reported in the cell wall of the more genetically tractable C. albicans relative, P. pastoris. Here we present the identification, cloning, and characterization of a novel family of fungal genes involved in beta-mannose transfer. Employing in silico analysis, we identified a family of four related new genes in P. pastoris and subsequently nine homologs in C. albicans. Biochemical, immunological, and structural analyses following deletion of four genes in P. pastoris and deletion of four genes acting specifically on C. albicans mannan demonstrated the involvement of these new genes in beta-1,2-oligomannoside synthesis. Phenotypic characterization of the strains deleted in beta-mannosyltransferase genes (BMTs) allowed us to describe the stepwise activity of Bmtps and acceptor specificity. For C. albicans, despite structural similarities between mannan and phospholipomannan, phospholipomannan beta-mannosylation was not affected by any of the CaBMT1-4 deletions. Surprisingly, depletion in mannan major beta-1,2-oligomannoside epitopes had little impact on cell wall surface beta-1,2-oligomannoside antigenic expression. 相似文献
12.
A glycosyltransferase was fused to the yeast cell wall protein Pir, which forms the Pir1-4 protein family and is incorporated into the cell wall by an unknown linkage to be displayed at the yeast cell surface. We first expressed the PIR1-HA-gma12+ fusion, in which gma12+ encodes alpha-1,2-galactosyltransferase from the fission yeast Schizosaccharomyces pombe under the Saccharomyces cerevisiae GAPDH promoter. The alpha-1,2-galactosyltransferase activity was detected at the surface of the intact cells that produce Pir1-HA-Gma12 fusion. To further demonstrate sequential oligosaccharide synthesis, two plasmids containing PIR1-HA-KRE2 and PIR2-FLAG-MNN1 fusion genes were constructed in which KRE2 and MNN1 encode alpha-1,2-mannosyltransferase and alpha-1,3-mannosyltransferase from S. cerevisiae, respectively. The intact yeast cells transformed with these two plasmids added mannoses initially with an alpha-1,2 linkage and subsequently with an alpha-1,3 linkage to the alpha-1,2-mannobiose acceptor in the presence of a GDP-mannose donor, demonstrating that Pir1 and Pir2 can be used as anchors to simultaneously immobilize several glycosyltransferases at the yeast cell surface. Based on the high acceptor specificity of glycosyltransferases, we propose a simple in vitro method for oligosaccharide synthesis using the yeast intact cell as a biocatalyst. 相似文献
13.
We have characterized a UDP-GlcNAc:Gal beta-3-GalNAc (GlcNAc----GalNAc) beta-6-N-acetylglucosaminyltransferase from rabbit small intestinal epithelium by using freezing point depression glycoprotein as the acceptor. Optimal enzyme activity was obtained at pH 7.0-7.5, at 3 mM MnCl2, and at 0.08% Triton X-100. Ca2+, Mg2+, and Ba2+ also enhanced enzyme activity. The apparent Michaelis constant was 4.80 mM for freezing point depression glycoprotein, 0.59 mM for periodate-treated porcine submaxillary mucin, 0.49 mM for Gal beta 1----3 GalNAc alpha Ph, and 1.03 mM for UDP-GlcNAc. No enzyme activity was observed when asialo ovine submaxillary mucin was used as the acceptor. The 14C-labeled oligosaccharide obtained by alkaline borohydride treatment of the product was shown to be a homogeneous trisaccharide by compositional analysis, Bio-Gel P-4 gel filtration, and high-performance liquid chromatography. The structure of the trisaccharide was identified as Gal beta 1----3-(GlcNAc beta 1----6)GalNAc-H2 by (a) identification of 2,3,4,6-tetramethyl-1,5-diacetylgalactitol and 1,4,5-trimethyl-3,6-diacetyl-2-N-methylacetamidogalactitol by gas-liquid chromatography-mass spectrometry and (b) the complete cleavage of the newly formed glycosidic bond by jack bean beta-hexosaminidase. The structure of the trisaccharide was confirmed by 1H nuclear magnetic resonance (270 MHz) and also by periodate oxidation of the trisaccharide followed by NaBH4 reduction, 4 N HCl hydrolysis, a second NaBH4 reduction, and the identification of threosaminitol on an amino acid analyzer. By acceptor competition studies, the enzyme activity was shown to be a much N-acetylglucosaminyltransferase. We postulate that this glycosyltransferase may play a key role in the regulation of mucin oligosaccharide synthesis. 相似文献
14.
A synthesis of alpha-series ganglioside GM1alpha (III(6)Neu5AcGgOse4Cer) containing C20-sphingosine(d20:1) is described. Glycosylation of 2-(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside with the glucosamine donor ethyl 3-O-acetyl-2-deoxy-4,6-O-[(4-methoxyphenyl)methylene]-2-phthalimido-1-thio-beta-D-glucopyranoside furnished a beta-(1-->4)-linked trisaccharide. Reductive cleavage of the p-methoxybenzylidene group followed by intramolecular inversion of its triflate afforded the desired trisaccharide, which was transformed into a trisaccharide acceptor via removal of the phthaloyl and O-acetyl groups followed by N-acetylation. A tetrasaccharide acceptor was obtained by glycosylation of the trisaccharide acceptor with dodecyl 2,3,4,6-tetra-O-benzoyl-1-thio-beta-D-galactopyranoside, followed by removal of the p-methoxybenzyl group. Coupling of the tetrasaccharide acceptor with ethyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-1-thio-5-trichloroacetamido-D-glycero-D-galacto-2-nonulopyranosid)onate and subsequent radical reduction gave the desired GM1alpha saccharide derivative, which was coupled with (2S,3R,4E)-2-azido-3-O-benzoyl-4-eicosene-1,3-diol after conversion into the imidate. 相似文献
15.
Enzymatic synthesis was attempted of six trisaccharides and 14 tetrasaccharides comprising beta-(1-->4)-linked D-glucose and D-xylose residues, using cellodextrin phosphorylase (CDP, EC 2.4.1.49) as the enzyme catalyst, with alpha-D-glucose 1-phosphate (1) or alpha-D-xylose 1-phosphate (2) as the donor substrates, and cellobiose (3), xylobiose (4), betaGlc-(1-->4)-Xyl (5), or betaXyl-(1-->4)-Glc (6) as the acceptor substrates. All enzymatic reactions were performed at pH 7.0 and the products purified by gel-filtration chromatography. We successfully synthesized all six hetero-trisaccharides and 10 of the 14 possible hetero-tetrasaccharides. It was not found possible to synthesize the four tetrasaccharides with a Xyl-->Glc sequence at their non-reducing ends employing this method. The stereochemistries of the isolated products were assessed by analysis of their 2D NMR spectra (DQF-COSY, TOCSY, HSQC, HMBC), confirming that all of the glycosidic bonds in the products were beta-(1-->4) linkages. 相似文献
16.
The nonnucleophilic mutant E383A beta-glucosidase from Streptomyces sp. has proven to be an efficient glycosynthase enzyme, catalyzing the condensation of alpha-glucosyl and alpha-galactosyl fluoride donors to a variety of acceptors. The enzyme has maximal activity at 45 degrees C, and a pH-dependence reflecting general base catalysis with an apparent kinetic pKa of 7.2. The regioselectivity of the new glycosidic linkage depends unexpectedly on the acceptor substrate. With aryl monosaccharide acceptors, beta-(1-->3) disaccharides are obtained in good to excellent yields, thus expanding the synthetic products available with current exo-glycosynthases. With xylopyranosyl acceptor, regioselectivity is poorer and results in the formation of a mixture of beta-(1-->3) and beta-(1-->4) linkages. In contrast, disaccharide acceptors produce exclusively beta-(1-->4) linkages. Therefore, the presence of a glycosyl unit in subsite +II redirects regioselectivity from beta-(1-->3) to beta-(1-->4). To improve operational performance, the E383A mutant was immobilized on a Ni2+-chelating Sepharose resin. Immobilization did not increase stability to pH and organic solvents, but the operational stability and storage stability were clearly enhanced for recycling and scaling-up. 相似文献
17.
The effect of substitution of the HO-6 of D-galactose with fluorine on the ability of alpha-(1-->3)-galactosyltransferase (EC 2.4.1.151) and beta-(1-->4)-galactosyltransferase (EC 2.4.1.22) to catalyze its transfer from UDP to an appropriate acceptor was determined. HPLC analyses indicated that each transferase properly catalyzed formation of the expected product [beta-D-Gal-(1-->4)-D-GlcNAc] for the beta-(1-->4)-galactosyltransferase and alpha-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-D-GlcNAc for the alpha-(1-->3)-D-galactosyltransferase] when UDP-alpha-D-Gal was the substrate. When UDP-6-deoxy-6-fluoro-alpha-D-galactose (6) was used in conjunction with each transferase, no product indicative of transfer of 6-deoxy-6-fluoro-D-galactose to its respective acceptor sugar was identified. 6-Deoxy-6-fluoro-D-galactose (3) was obtained by hydrolysis of methyl 6-deoxy-6-fluoro-alpha-D-galactopyranoside, synthesized by the selective fluorination of methyl alpha-D-galactopyranoside with diethylaminosulfur trifluoride (DAST), with aqueous trifluoroacetic acid. Acetylation of 3 gave crystalline 1,2,3,4-tetra-O-acetyl-6-deoxy-6-fluoro-beta-D-galactopyranose, which was converted to the corresponding 1-alpha-phosphate and used for the synthesis of 6. 相似文献
18.
A practical synthesis of beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Xyl-(1-->OMe) was achieved by coupling of methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate trichloroacetimidate with a trisaccharide acceptor. The trisaccharide acceptor was obtained by condensation of 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate with methyl 2,3-di-O-benzoyl-beta-D-xylopyranoside, followed by deallylation. The beta-(1-->3)-linked disaccharide was prepared readily with p-methoxyphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranoside as the key synthon. The alpha-(1-->3)-linkage was formed in considerable amount with galactose mono- and disaccharide trichloroacetimidate donors with C-2 neighboring group participation. 相似文献
19.
Human blood group O plasma was found to contain an N-acetylgalactosaminyltransferase which catalyzes the transfer of N-acetylgalactosamine from UDP-GalNAc to Gal beta 1-->4Glc, Gal beta 1-->4GlcNAc, asialo-alpha 1-acid glycoprotein, and Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-ceramide, but not to Gal beta 1-->3GlcNAc. The enzyme required Mn2+ for its activity and showed a pH optimum at 7.0. The reaction products were readily hydrolyzed by beta-N-acetylhexosaminidase and released N-acetylgalactosamine. Apparent Km values for UDP-GalNAc, Mn2+, lactose, N-acetyllactosamine, and terminal N-acetyllactosaminyl residues of asialo-alpha 1-acid glycoprotein were 0.64, 0.28, 69, 20, and 1.5 mM, respectively. Studies on acceptor substrate competition indicated that all the acceptor substrates mentioned above compete for one enzyme, whereas the enzyme can be distinguished from an NeuAc alpha 2-->3Gal beta-1,4-N-acetylgalactosaminyltransferase, which also occurs in human plasma. The methylation study of the product formed by the transfer of N-acetylgalactosamine to lactose revealed that N-acetylgalactosamine had been transferred to the carbon-3 position of the beta-galactosyl residue. Although the GalNAc beta 1-->3Gal structure is known to have the blood group P antigen activity, human plasma showed no detectable activity of Gal alpha 1-->4Gal beta-1,3-N-acetylgalactosaminyltransferase, which is involved in the synthesis of the major P antigen-active glycolipid, GalNAc beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc-ceramide. Hence, the GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure is synthesized by the novel Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylgalactosaminyltransferase. 相似文献
20.
Malissard M Zeng S Berger EG 《Biochemical and biophysical research communications》2000,267(1):169-173
The cDNAs encoding soluble forms of human beta-1, 4-galactosyltransferase I (EC 2.4.1.22), alpha-2,6-sialyltransferase (EC 2.4.99.1), and alpha-1,3-fucosyltransferase VI (EC 2.4.1.65), respectively, have been expressed in the methylotrophic yeast Pichia pastoris. The vector pPIC9 was used, which contains the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway. The recombinant enzymes had similar kinetic properties as their native counterparts. Their identity was confirmed by Western blotting. Recombinant enzymes may be used for in vitro synthesis of oligosaccharides. 相似文献