Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants.
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.
Sugiura
Nobuo
N
Institute for Molecular Science of Medicine, Aichi Medical University, Yazako, Nagakute, Aichi 480-1195, Japan. nsugiura@aichi-med-u.ac.jp
Shimokata
Satoshi
S
Minamisawa
Toshikazu
T
Hirabayashi
Jun
J
Kimata
Koji
K
Watanabe
Hideto
H
D015153:Blotting, Western; D002240:Carbohydrate Sequence; D020134:Catalytic Domain / Q000235:genetics; D002807:Chondroitin / Q000096:biosynthesis* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized / Q000235:genetics*; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D017354:Point Mutation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D004800:Enzymes, Immobilized; D002807:Chondroitin; D006602:Hexosyltransferases; C460368:chondroitin polymerase, E coli