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

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 SN2-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.

Expression, purification and biochemical characterization of AtFUT1, a xyloglucan-specific fucosyltransferase from Arabidopsis thaliana.

Efforts to identify genes and characterize enzymes involved in the biosynthesis of plant cell wall polysaccharides have yet to produce and purify to homogeneity an active plant cell wall synthesizing enzyme suitable for structural studies. In Arabidopsis, the last step of xyloglucan (XG) biosynthesis is catalyzed by fucosyltransferase 1 (AtFUT1), which transfers l-fucose from GDP-ß-l-fucose to a specific galactose on the XG core. Here, we describe the production of a soluble form of AtFUT1 (HisΔ68-AtFUT1) and its purification to milligram quantities. An active form of AtFUT1 was produced in an insect cell culture medium, using a large-scale expression system, and purified in a two-step protocol. Characterization of purified HisΔ68-AtFUT1 revealed that the enzyme behaves as a non-covalent homodimer in solution. A bioluminescent transferase assay confirmed HisΔ68-AtFUT1 activity on its substrates, namely GDP-fucose and tamarind XG, with calculated Km values of 42 µM and 0.31 µM, respectively. Moreover, the length of the XG-derived acceptor quantitatively affected fucosyltransferase activity in a size-dependent manner. The affinity of HisΔ68-AtFUT1 for tamarind XG and GDP was determined using isothermal titration calorimetry (ITC). Interestingly, ITC data suggest that HisΔ68-AtFUT1 undergoes conformational changes in the presence of its first co-substrate (XG or GDP), which then confers greater affinity for the second co-substrate. The procedure described in this study can potentially be transferred to other enzymes involved in plant cell wall synthesis.

The galactoside 2-α-L-fucosyltransferase FUT1 from Arabidopsis thaliana: crystallization and experimental MAD phasing.

The plant cell wall is a complex network of polysaccharides made up of cellulose, hemicelluloses and pectins. Xyloglucan (XyG), which is the main hemicellulosic component of dicotyledonous plants, has attracted much attention for its role in plant development and for its many industrial applications. The XyG-specific fucosyltransferase (FUT1) adds a fucose residue from GDP-fucose to the 2-O position of the terminal galactosyl residues on XyG side chains. Recombinant FUT1 from Arabidopsis thaliana was crystallized in two different crystal forms, with the best diffracting crystals (up to 1.95 Šresolution) belonging to the monoclinic space group P21, with unit-cell parameters a = 87.6, b = 84.5, c = 150.3 Å, ß = 96.3°. Ab initio phases were determined using a two-wavelength anomalous dispersion experiment on a tantalum bromide-derivatized crystal with data collected at the rising and descending inflection points of the Ta white line. An interpretable electron-density map was obtained after elaborate density modification. Model completion and structural analysis are currently under way.

Unique features of a Pseudomonas aeruginosa α2-macroglobulin homolog.

UNLABELLED: Human pathogens frequently use protein mimicry to manipulate host cells in order to promote their survival. Here we show that the opportunistic pathogen Pseudomonas aeruginosa synthesizes a structural homolog of the human α2-macroglobulin, a large-spectrum protease inhibitor and important player of innate immunity. Small-angle X-ray scattering analysis demonstrated that the fold of P. aeruginosa MagD (PA4489) is similar to that of the human macroglobulin and undergoes a conformational modification upon binding of human neutrophil elastase. MagD synthesis is under the control of a general virulence regulatory pathway including the inner membrane sensor RetS and the RNA-binding protein RsmA, and MagD undergoes cleavage from a 165-kDa to a 100-kDa form in all clinical isolates tested. Fractionation and immunoprecipitation experiments showed that MagD is translocated to the bacterial periplasm and resides within the inner membrane in a complex with three other molecular partners, MagA, MagB, and MagF, all of them encoded by the same six-gene genetic element. Inactivation of the whole 10-kb operon on the PAO1 genome resulted in mislocalization of uncleaved, in trans-provided MagD as well as its rapid degradation. Thus, pathogenic bacteria have acquired a homolog of human macroglobulin that plays roles in host-pathogen interactions potentially through recognition of host proteases and/or antimicrobial peptides; it is thus essential for bacterial defense. IMPORTANCE: The pathogenesis of Pseudomonas aeruginosa is multifactorial and relies on surface-associated and secreted proteins with different toxic activities. Here we show that the bacterium synthesizes a 160-kDa structural homolog of the human large-spectrum protease inhibitor α2-macroglobulin. The bacterial protein is localized in the periplasm and is associated with the inner membrane through the formation of a multimolecular complex. Its synthesis is coregulated at the posttranscriptional level with other virulence determinants, suggesting that it has a role in bacterial pathogenicity and/or in defense against the host immune system. Thus, this new P. aeruginosa macromolecular complex may represent a future target for antibacterial developments.