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| Content Provider | Springer Nature Link |
|---|---|
| Author | Sugiura, buo Shimokata, Satoshi Minamisawa, Toshikazu Hirabayashi, Jun Kimata, Koji Watanabe, Hideto |
| Copyright Year | 2008 |
| Abstract | Escherichia coli strain K4 expresses a chondroitin (CH)-polymerizing enzyme (K4CP) that contains two glycosyltransferase active domains. K4CP alternately transfers glucuronic acid (GlcA) and N-acetyl-galactosamine (GalNAc) residues using UDP-GlcA and UDP-GalNAc donors to the nonreducing end of a CH chain acceptor. Here we generated two K4CP point mutants substituted at the UDP-sugar binding motif (DXD) in the glycosyltransferase active domains, which showed either glycosyltransferase activity of the intact domain and retained comparable activity after immobilization onto agarose beads. The mutant enzyme-immobilized beads exhibited an addition of GlcA or GalNAc to GalNAc or GlcA residue at the nonreducing end of CH oligosaccharides and sequentially elongated pyridylamine-conjugated CH (PA-CH) chain by the alternate use. The sequential elongation up to 16-mer was successfully achieved as assessed by fluorescent detection on a gel filtration chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI potential lift tandem TOF mass spectrometry (MALDI-LIFT-TOF/TOF MS/MS) analyses in the negative reflection mode. This method provides exactly defined CH oligosaccharide derivatives, which are useful for studies on glycosaminoglycan functions. |
| Starting Page | 521 |
| Ending Page | 530 |
| Page Count | 10 |
| File Format | |
| ISSN | 02820080 |
| Journal | Glycoconjugate Journal |
| Volume Number | 25 |
| Issue Number | 6 |
| e-ISSN | 15734986 |
| Language | English |
| Publisher | Springer US |
| Publisher Date | 2008-02-05 |
| Publisher Place | Boston |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Chondroitin Stepwise glycosylation MALDI-TOF MS MS/MS fragmentation Pyridylamination Pathology Biochemistry |
| Content Type | Text |
| Resource Type | Article |
| Subject | Cell Biology Biochemistry Molecular Biology |
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