The nodZ gene, which is present in various rhizobial species, is involved in the addition of a fucose residue in an ␣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.The symbiosis between rhizobial species and leguminous plants, resulting in the formation of nitrogen-fixing root nodules, is a species-specific process that is mediated by signal molecules from both the plant and the bacterium. During the initial phases of nodulation (root hair curling and bacterial entry), flavonoids secreted by the host plant induce an activation of nod genes that are involved in the synthesis of lipochitin oligosaccharides, called Nod factors. Once Nod factors have opened the root hair door to the invading rhizobia, additional bacterial signals appear necessary for continued development of the infection (for recent reviews, see references 9, 41, and 51). The basic Nod factors comprise a chitin backbone formed by the assembly of a few 1,4-linked N-acetyl-Dglucosamine (GlcNAc) residues and an acyl chain attached at the nonreducing end (16). The nature of the fatty acyl chain,