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Results for “Hexosyltransferases”

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At least 19 recordsLinked to original sources
PubMed1984

The extraction and mechanism of a novel isomaltulose-synthesizing enzyme from Erwinia rhapontici.

The single enzyme that mediates the bioconversion is demonstrated to be located in the cells' periplasmic space, a site that facilitates its use as an industrial biocatalyst, and to be a previously undescribed hexosyltransferase with four novel features. The enzyme is sucrose-specific, and has an intramolecular mechanism in which both glucose and fructose residues appear to be enzyme-bound. Thirdly, it is reaction-non-selective, forming simultaneously isomaltulose and a second hitherto uncharacterized alpha-(1----1)-linked disaccharide (trehalulose), by hydrolysis of sucrose followed by reaction of glucose with the C-6 and C-1 positions of the fructofuranose respectively. Finally, on extended incubation an unusual recycling mechanism caused the concentration of isomaltulose, the kinetically preferred product, to reach a transient maximum concentration and then fall, and the concentration of trehalulose, the thermodynamically favoured product, to rise slowly.

Carbohydrate Epimerases↗
PubMed1998

Identification of the active site serine of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by electrospray mass spectrometry.

Penicillin-binding protein 2a (PBP2a), a high molecular mass PBP, is the primary enzyme responsible for the beta-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). Inhibition of a PBP such as PBP2a by beta-lactams is due to covalent modification of an active site serine residue. Based on the sequence alignment with well studied beta-lactamases, DD-carboxypeptidases and other high molecular mass PBPs, the serine of a tetrad S403XXK in PBP2a was tentatively identified as the penicillin-binding site. However, direct evidence for the involvement of serine403 has not been reported. In this study, a method which combines liquid chromatography/electrospray mass spectrometry (LC/MS) and nano-electrospray MS for the identification of the active site serine in PBP2a is described. The covalent binding of the beta-lactams was carried out in vitro with the recombinant PBP2a. Peptide mapping of the cyanogen bromide fragments from penicilloyl-PBP2a, using microbore LC/MS, provided a rapid identification of the modified peptide with a 334 Da mass increase. The acylated peptide was isolated and further digested with trypsin. Nano-electrospray MS/MS sequencing of the acylated peptide in the tryptic digest showed that the penicillin was indeed attached to serine403.

Acylation↗
PubMed1999

Structural characterization of Acetobacter diazotropicus levansucrase by matrix-assisted laser desorption/ionization mass spectrometry: identification of an N-terminal blocking group and a free-thiol cysteine residue.

High-resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize the primary structure of the levansucrase (EC 2.4.1.10) secreted by Acetobacter diazotropicus SRT4. The technique permitted not only the reading frame of this enzyme, the amino acid sequence of which was deduced from DNA, but also the elucidation of an N-terminal blocking group and the position of a disulfide bridge between Cys309 and Cys365 among the three Cys residues. A free cysteine (Cys127) was identified by modifying an intact molecule with a sulfhydryl reagent, 5-(octyldithio)-2-nitrobenzoic acid, under non-reducing conditions. In addition, the enzyme obtained by site-directed mutagenesis at Asp279 to Asn279 was also identified by the above methods. Post-source decay analysis of the tryptic peptide containing the mutation site unequivocally revealed an Asn residue at position 279.

Acetobacter↗
PubMed1996

The targeting of Bacillus subtilis levansucrase in yeast is correlated to both the hydrophobicity of the signal peptide and the net charge of the N-terminus mature part.

We compared the ability of signal sequences from various Bacillus or yeast secreted proteins to direct Bacillus subtilis levansucrase into the secretion pathway of the yeast Saccharomyces cerevisiae. The efficiency of these sequences correlated with the overall hydrophobicity of their h-domain and was independent of their origin. Furthermore, the net charge of the proximal protein sequence downstream from the signal sequence contributed to the competence of the heterologous proteins to be secreted by yeast. Modification of this net charge allowed the protein to be translocated under the control of the yeast invertase signal sequence. Moreover, glycosylation of levansucrase did not modify significantly the fructosyl polymerase activity.

Acid Phosphatase↗
PubMed1999

Schizosaccharomyces pombe stt3+ is a functional homologue of Saccharomyces cerevisiae STT3 which regulates oligosaccharyltransferase activity.

The Saccharomyces cerevisiae STT3 (ScSTT3) gene encodes a protein which is involved in protein glycosylation via the regulation of oligosaccharyltransferase activity. We have cloned and isolated the Schizosaccharomyces pombe STT3 homologous gene (Spstt3+). The Spstt3+ gene encodes a protein consisting of 749 amino acid residues which has significant homology with ScStt3p and the mouse Stt3p-homologue Itm1p. Disruption of the Spstt3+ gene shows that this gene is essential for growth. Like Itm1, Spstt3+ partially suppressed the temperature sensitivity of the stt3-1 mutation of S. cerevisiae, indicating that Spstt3+ is a functional and structural homologue of the ScSTT3 gene.

Amino Acid Sequence↗
PubMed1998

Structural model for family 32 of glycosyl-hydrolase enzymes.

A structural model is presented for family 32 of the glycosyl-hydrolase enzymes based on the beta-propeller fold. The model is derived from the common prediction of two different threading methods, TOPITS and THREADER. In addition, we used a correlated mutation analysis and prediction of active-site residues to corroborate the proposed model. Physical techniques (circular dichroism and differential scanning calorimetry) confirmed two aspects of the prediction, the proposed all-beta fold and the multi-domain structure. The most reliable three-dimensional model was obtained using the structure of neuraminidase (1nscA) as template. The analysis of the position of the active site residues in this model is compatible with the catalytic mechanism proposed by Reddy and Maley (J. Biol. Chem. 271:13953-13958, 1996), which includes three conserved residues, Asp, Glu, and Cys. Based on this analysis, we propose the participation of one more conserved residue (Asp 162) in the catalytic mechanism. The model will facilitate further studies of the physical and biochemical characteristics of family 32 of the glycosyl-hydrolases.

Amino Acid Sequence↗
PubMed1978

Evaluation of two plasma fucosyltransferases as marker enzymes in non-Hodgkin's lymphoma.

Levels of two fusosyltransferases were measured in plasmas of patients with non-Hodgkin's lymphoma at different phases of the disease. The level of a GDP-fucose: galactoside fucosyltransferase (EC 2.4.1.69) was found elevated in nonresponding patients and was correlated with estimated tumor burden. Enzyme levels in the normal range were found in patients in remission, maintained on chemotherapy, or unmaintained. The plasma level of a GDP-fucose; N-acetylglucosaminide fucosyltransferase (EC 2.4.1.68) was elevated in all individuals receiving drug therapy regardless of diesease status, but returned to normal levels during unmaintained remissions.

Acetylglucosamine↗
PubMed1983

Serum sialyltransferase and fucosyltransferase activities in patients with bronchial carcinoma.

Serum sialyltransferases and fucosyltransferases measured by an affinity adsorbent technique were studied in 27 exactly defined patients with malignant pulmonary diseases. Fourteen patients with benign pulmonary diseases and 56 with benign surgical diseases were used as controls. Enzyme activities were expressed as amounts of labeled precursor molecules incorporated into endogenous acceptors in counts per minute (cpm). The mean sialyltransferase activity was 583 cpm in bronchial carcinoma, 485 cpm in benign pulmonary disease and 428 cpm in benign surgical disease. The only statistically significant difference was between bronchial carcinoma and benign surgical disease. The mean fucosyltransferase activity was 813 cpm in bronchial carcinoma, 436 cpm in benign pulmonary disease and 255 cpm in benign surgical disease. All the differences were statistically significant. There were no statistically significant differences between the WHO histologic bronchial carcinoma groups. The correlation between sialyltransferase and fucosyltransferase activity in bronchial carcinoma was statistically significant (r = 0.59). In squamous cell carcinoma (N = 6), it was strongly significant (r = 0.96) and there was a significant correlation also in small cell carcinoma (N = 10; r = 0.79) but not in adenocarcinoma (N = 9; r = 0.30) and benign pulmonary disease (N = 14; r = 0.44). It is suggested that serum sialyltransferases and fucosyl transferases would not be decisive for diagnosis when used alone in bronchial carcinoma, but could be included in a screening test battery.

Bronchial Neoplasms↗
PubMed1988

Cancer-associated elevation of alpha(1----3)-L-fucosyltransferase activity in human serum.

GDP-fucose:N-acetylglucosaminide alpha(1----3)-L-fucosyltransferase activity was measured in sera of patients with various cancers using a synthetic substrate, N-acetyl-2'-O-methyllactosamine, as an acceptor. One hundred twenty-four of the 169 patients showed significantly high levels of the enzyme activity when compared to healthy controls, irrespective of the location of their tumor. However, enzyme levels were in the normal range in patients with non-neoplastic diseases, such as infectious disease, liver disease, and other inflammatory problems as well as in leukemic patients. The chromatofocusing profile of the enzyme using PBE-94 gel over a pH gradient from pH 6.0 to 4.0 demonstrated that the level of enzyme eluted at pH 5.4 was markedly elevated in the sera of stomach and ovarian cancer patients. A correlation was established between alpha(1----3)-L-fucosyltransferase activity and the presence of malignancy which may be used to evaluate the utility of the enzyme as a tumor marker.

Biomarkers, Tumor↗
PubMed1989

Serum alpha(1----3)-L-fucosyltransferase, carcinoembryonic antigen, and sialyl Lewis X-i antigen levels in lung cancer.

Serum alpha(1----3)-L-fucosyltransferase activity was measured in 58 patients with lung cancer, 27 benign diseases, and in 100 healthy controls. The levels of enzyme activity were significantly higher in the sera of patients with cancer when compared to those in benign diseases and healthy controls. The elevation of the enzyme activity correlated with the clinical stages and to the size of the primary tumors. Follow-up studies with various stages showed that the enzyme activity was useful in tracking the clinical course of disease after surgery. To evaluate the usefulness of this enzyme as a diagnostic marker, carcinoembryonic antigen (CEA) and sialyl Lewis X-i antigen levels were also measured. The results indicate that alpha(1----3)-L-fucosyltransferase could be a more specific tumor marker than such tumor-associated antigens in lung cancer.

Carcinoembryonic Antigen↗
PubMed2002

Moenomycin-mediated affinity purification of penicillin-binding protein 1b.

The antibiotic moenomycin A inhibits the biosynthesis of peptidoglycan, the main structural polymer of the bacterial cell wall. The inhibition is based on a reversible binding of the antibiotic to one of the substrate binding sites at enzymes such as the penicillin binding protein 1b (PBP 1b). This binding has been employed to isolate PBP 1b by affinity chromatography. Suitable ligands have been prepared from moenomycin A and coupled both to affinity supports and to surface plasmon resonance sensor surfaces. The reactions that take place upon immobilization of the ligands to the affinity support and the sensor surface, respectively, have been studied in detail. With the help of surface plasmon resonance the optimal conditions for binding of PBP 1b to moenomycin-derivated ligands have been established. For the first time the selective binding of the moenomycin sugar moiety to the enzyme has been demonstrated.

Bacterial Proteins↗
PubMed2002

A surface plasmon resonance analysis of the interaction between the antibiotic moenomycin A and penicillin-binding protein 1b.

The antibiotic moenomycin A inhibits the biosynthesis of peptidoglycan, the main structural polymer of the bacterial cell wall. The inhibition is based on a reversible binding of the antibiotic to one of the substrate binding sites in enzymes such as penicillin-binding protein (PBP) 1b. A novel assay based on surface plasmon resonance (SPR) has been established that can be used to investigate selective binding of the moenomycin sugar moiety and other transglycosylase inhibitors to this enzyme. Suitable ligands were prepared from moenomycin A and coupled to SPR sensor surfaces. Moenomycin analogues with structural variations were used to perform competitive SPR experiments with PBP 1b. The SPR results confirm for the first time that the trisaccharide fragment of moenomycin A (C-E-F-G-H-I) is the minimal structure that possesses all moieties sufficient for biological activity and for affinity towards PBP 1b. The method seems to be appropriate for use in screens for transglycosylase inhibitors that bind to the moenomycin-binding site of the enzyme.

Bacterial Proteins↗
PubMed2001

Fluorescent Bocillins: synthesis and application in the detection of penicillin-binding proteins.

Novel fluorescent analogs of penicillin V were synthesized and evaluated for efficacy in the detection of penicillin binding proteins (PBPs). These molecules include the full structure of penicillin V, with the potent Bodipy fluorophore attached to the para-position of the penicillin V phenyl group. The green fluorescent Bocillin FL and the near-infrared (IR) fluorescent Bocillin 650/665 probes were shown to bind to PBPs, both purified and from membrane preparations, with high affinity and specificity. These reagents allow for facile detection of 2-4 ng of purified PBP with the aid of a fluorescent scanner.

Bacteria↗