Bacterial nodulation protein NodZ is a chitin oligosaccharide fucosyltransferase which can also recognize related substrates of animal origin.
The nodZ gene, which is present in various soil bacteria such as Bradyrhizobium japonicum, Azorhizobium caulinodans, and Rhizobium loti, is involved in the addition of a fucosyl residue to the reducing N-acetylglucosamine residue of lipochitin oligosaccharide (LCO) signal molecules. Using an Escherichia coli strain that produces large quantities of the NodZ protein of B. japonicum, we have purified the NodZ protein to homogeneity. The purified NodZ protein appears to be active in an in vitro transfucosylation assay in which GDP-beta-fucose and LCOs or chitin oligosaccharides are used as substrates. The products of the in vitro reaction using chitin oligosaccharides as substrate were studied by using mass spectrometry, linkage analysis, and composition analysis. The data show that one fucose residue is added to C6 of the reducing-terminal N-acetylglucosamine residue. The substrate specificity of NodZ protein was analyzed in further detail, using radiolabeled GDP-beta-fucose as the donor. The results show that chitin oligosaccharides are much better substrates than LCOs, suggesting that in Rhizobium NodZ fucosylates chitin oligosaccharides prior to their acylation. The free glycan core pentasaccharides of N-linked glycoproteins are also substrates for NodZ. Therefore, the NodZ enzyme seems to have an activity equivalent to that of the enzyme involved in the addition of the C6-linked fucosyl substituent in the glycan core of N-linked glycoproteins in eukaryotes. Oligosaccharides that contain only one N-acetylglucosamine at the reducing terminus are also substrates for NodZ, although in this case very high concentrations of such oligosaccharides are needed. An example is the leukocyte antigen Lewis-X, which can be converted by NodZ to a novel fucosylated derivative that could be used for binding studies with E-selectin.
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Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca Morelos 62271, Mexico.
Wijfjes
A H
AH
Bloemberg
G V
GV
Blok-Tip
L
L
López-Lara
I M
IM
Lugtenberg
B J
BJ
Thomas-Oates
J E
JE
Spaink
H P
HP
D000117:Acetylglucosamine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002686:Chitin / Q000378:metabolism*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D002686:Chitin; D006154:Guanosine Diphosphate Fucose; D005647:Fucosyltransferases; C103941:NodZ protein, bacteria; D000117:Acetylglucosamine