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Journal of Bacteriology, December 2001, p. 7067-7075, Vol. 183, No. 24
Centre de Recherches sur les
Macromolécules Végétales and Joseph Fourier
University, CNRS, Grenoble, France
Received 11 June 2001/Accepted 24 September 2001
The nodZ gene, which is present in various rhizobial
species, is involved in the addition of a fucose residue in an
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7067-7075.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification of Essential Amino Acids in the
Azorhizobium caulinodans Fucosyltransferase
NodZ
1-6
linkage to the reducing N-acetylglucosamine residue of
lipo-chitin oligosaccharide signal molecules, the so-called Nod
factors. Fucosylation of Nod factors is known to affect nodulation
efficiency and host specificity. Despite a lack of overall sequence
identity, NodZ proteins share conserved peptide motifs with mammalian
and plant fucosyltransferases that participate in the biosynthesis of
complex glycans and polysaccharides. These peptide motifs are thought
to play important roles in catalysis. NodZ was expressed as an active
and soluble form in Escherichia coli and was subjected
to site-directed mutagenesis to investigate the role of the most
conserved residues. Enzyme assays demonstrate that the replacement of
the invariant Arg-182 by either alanine, lysine, or aspartate results
in products with no detectable activity. A similar result is obtained
with the replacement of the conserved acidic position (Asp-275) into
its corresponding amide form. The residues His-183 and Asn-185 appear
to fulfill functions that are more specific to the NodZ subfamily.
Secondary structure predictions and threading analyses suggest the
presence of a "Rossmann-type" nucleotide binding domain in the half
C-terminal part of the catalytic domain of fucosyltransferases.
Site-directed mutagenesis combined with theoretical approaches have
shed light on the possible nucleotide donor recognition mode for NodZ
and related fucosyltransferases.
*
Corresponding author. Mailing address: CERMAV-CNRS, BP
53, F-38041 Grenoble Cedex 09, France. Phone: (33) (0) 4-7603-7635. Fax: (33) (0) 4-7654-7203. E-mail:
Christelle.Breton{at}cermav.cnrs.fr.
Dedicated to the memory of André Verbert.
Present address: Department of Biochemistry II, Kagoshima
University School of Medicine, Kagoshima, Japan.
§
Present address: UJF-CNRS FRE 2029, CERMO, Grenoble, France.
Journal of Bacteriology, December 2001, p. 7067-7075, Vol. 183, No. 24
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7067-7075.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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