Identification of essential amino acids in the Azorhizobium caulinodans fucosyltransferase NodZ.
The nodZ gene, which is present in various rhizobial species, is involved in the addition of a fucose residue in an alpha 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.
Chazalet
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V
Centre de Recherches sur les Macromolécules Végétales and Joseph Fourier University, CNRS, Grenoble, France.
Uehara
K
K
Geremia
R A
RA
Breton
C
C
D000595:Amino Acid Sequence; D020599:Azorhizobium caulinodans / Q000201:enzymology*; D001426:Bacterial Proteins; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005647:Fucosyltransferases; C103941:NodZ protein, bacteria