Identification of Essential Amino Acids in theAzorhizobium caulinodansFucosyltransferase NodZ

Abstract

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,

Chaperone Activity of Protein O -Fucosyltransferase 1 Promotes Notch Receptor Folding

“…The first R in this motif is invariant among all three fucosyltransferase families (Fig. 4A) and is essential for the activity of a1,6-and a1,2-fucosyltransferases in vitro and in vivo (18,19,21). We mutated this R in OFUT1, making OFUT1 R245K and OFUT1 R245A .…”

“…Because the ER lacks detectable GDP-fucose transporter activity, we thought that OFUT1 might facilitate Notch folding independently of its fucosyltransferase activity. A comparative study of sequences from a1,2-fucosyltransferases and a1,6-fucosyltransferases identified a GXHXR(R/H) motif that is implicated in donor substrate binding (18,19); O-fucosyltransferases include a related motif (20). The first R in this motif is invariant among all three fucosyltransferase families (Fig.…”

Characterization of WbiQ: An α1,2-fucosyltransferase from Escherichia coli O127:K63(B8), and synthesis of H-type 3 blood group antigen

“…Of these WbsJ and α1,2-FucT from H. pylori have unique substrate specificities and have demonstrated applicability in the synthesis of relevant fucose containing oligosaccharides [14, 16]. Regardless of the species that the α1,2- FucT was cloned from, no structural information is yet available for the α1,2-FucT subfamily (unlike the evolutionary related α1,6-FucT subfamily) [17]. As such, our understanding of the α1,2-FucT mechanism and roles of specific amino acid motifs are limited and are based off the available α1,6-FucT and α1,3-FucT crystal structures.…”

Identification of Putative Rhamnogalacturonan-II Specific Glycosyltransferases in Arabidopsis Using a Combination of Bioinformatics Approaches

“…The known α3-FucTs and α4-FucTs classify in a different GT family (GT10) not included in the FucT superfamily. This superfamily is characterized by the presence of three conserved peptide regions (called motifs I, II, and III) [14],[64],[75] (Fig. 5).…”