Biochemical characterization of a new β-1,3-galactosyltransferase WbuP from Escherichia coli O114 that catalyzes the second step in O-antigen repeating-unit.
In this study, synthetic acceptor substrate GlcNAc alpha-PO3-PO3-(CH2)11-O-phenyl (GlcNAc-PP-PhU) was employed in glycosyl transferase assays to characterize the WbuP galactosyltransferase activity. This activity was time- and enzyme concentration-dependent. The optimal enzyme activity was observed at pH 6.5 and 25°C. The enzyme requires Mn(2+) ions for maximal activity and detergents in the assay did not increase glycosyltransfer activity. The enzyme was shown to be specific for the UDP-Gal donor substrate. Kinetic parameters were determined for UDP-Gal, and GlcNAc-PP-PhU. The enzyme product was determined to have a β-1,3-linkage using strategies based on exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as well as collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MS(n)). Our results conclusively demonstrate that the wbuP gene of Escherichia coli O114 encodes a UDP-Gal: GlcNAc α-pyrophosphate-lipid β-1,3-Gal-transferase that transfers the second sugar moiety in the assembly of the O114 repeating unit.
TEDA School of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin 300457, PR China. Electronic address: daweizhou@nankai.edu.cn.
Dawei
D
Zhou
Natalia
N
Utkina
Diange
D
Li
Chenying
C
Dong
Tatyana
T
Druzhinina
Vladimir
V
Veselovsky
Bin
B
Liu
D055162:Biocatalysis; D002236:Carbohydrate Conformation; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D019081:O Antigens / Q000737:chemistry / Q000378:metabolism*
D019081:O Antigens; D005700:Galactosyltransferases
Escherichia coli O-antigen; Functional characterization; Glycosyltransferase; Mass spectrometry