Structure of Arabidopsis thaliana FUT1 Reveals a Variant of the GT-B Class Fold and Provides Insight into Xyloglucan Fucosylation

Abstract

The plant cell wall is a complex and dynamic network made mostly of cellulose, hemicelluloses, and pectins. Xyloglucan, the major hemicellulosic component in Arabidopsis thaliana, is biosynthesized in the Golgi apparatus by a series of glycan synthases and glycosyltransferases before export to the wall. A better understanding of the xyloglucan biosynthetic machinery will give clues toward engineering plants with improved wall properties or designing novel xyloglucan-based biomaterials. The xyloglucan-specific α2-fucosyltransferase FUT1 catalyzes the transfer of fucose from GDP-fucose to terminal galactosyl residues on xyloglucan side chains. Here, we present crystal structures of Arabidopsis FUT1 in its apoform and in a ternary complex with GDP and a xylo-oligosaccharide acceptor (named XLLG). Although FUT1 is clearly a member of the large GT-B fold family, like other fucosyltransferases of known structures, it contains a variant of the GT-B fold. In particular, it includes an extra C-terminal region that is part of the acceptor binding site. Our crystal structures support previous findings that FUT1 behaves as a functional dimer. Mutational studies and structure comparison with other fucosyltransferases suggest that FUT1 uses a S2-like reaction mechanism similar to that of protein-O-fucosyltransferase 2. Thus, our results provide new insights into the mechanism of xyloglucan fucosylation in the Golgi.

Structural, mutagenic and in silico studies of xyloglucan fucosylation in Arabidopsis thaliana suggest a water‐mediated mechanism

“…() using insect‐cell cultures and those used by Rocha et al . () for structural analysis, where they were only able to achieve 4 mg L −1 after extensive optimization (Cicéron et al ., ; Rocha et al ., ). Expression of GFP‐ At FUT1 in the glycosylation mutant HEK293S (GnT‐) cells limited N ‐glycosylation to Man 5 GlcNAc 2 structures and allowed the cleavage of these glycans with endoglycosidase F1 (EndoF1), resulting in a single GlcNAc residue at the glycosylation site.…”

“…In a very recent study, Rocha et al . performed an independent structural characterization of the At FUT1 enzyme in complex with GDP, acceptor XLLG, and propose a plausible reaction mechanism (Rocha et al ., ). We compare and contrast our multi‐pronged research findings on At FUT1 to challenge the proposed mechanism wherein the amino acid residue Asp300 is suggested to act as a base for an S N 2‐catalyzed mechanism.…”

Monitoring Polysaccharide Dynamics in the Plant Cell Wall

“…Even though most of the wall Fuc is present in fucogalactoXyG (Zablackis et al, 1996), an alkynylated Fuc analog only labeled RG I (Anderson et al, 2012). Apparently, the corresponding XyG:fucosyltransferase is not able to accept the Fuc analog as a donor substrate (Perrin et al, 1999;Rocha et al, 2016), while the RG I:fucosyltransferase does. These experiments highlight one of the current limitations of using click chemistry or other sugar analogs to monitor wall dynamics.…”

Arabidopsis thaliana α1,2‐l‐fucosyltransferase catalyzes the transfer of l‐galactose to xyloglucan oligosaccharides

“…In this study, the effects of divalent cations were analyzed, and Mn 2+ was found to inhibit the L-galactosyltransferase activity of AtFUT1 as described for the L-fucosyltransferase activity of the Pisum sativum FUT1 enzyme [28]. The crystallographic observation of AtFUT1 revealed that it can be classified as a GT-B enzyme and that the GDP moiety is coordinated with AtFUT1 by water-mediated contacts, not by divalent cations as with a GT-A enzyme, which requires divalent cations for donor substrate interaction [26,29]. Mn 2+ is known to coordinate with the phosphodiester bonds of ADP and ATP [30].…”