title: Structures of NodZ α1,6-fucosyltransferase in complex with GDP and GDP-fucose.
abstract: Rhizobial NodZ α1,6-fucosyltransferase (α1,6-FucT) catalyzes the transfer of the fucose (Fuc) moiety from guanosine 5'-diphosphate-β-L-fucose to the reducing end of the chitin oligosaccharide core during Nod-factor (NF) biosynthesis. NF is a key signalling molecule required for successful symbiosis with a legume host for atmospheric nitrogen fixation. To date, only two α1,6-FucT structures have been determined, both without any donor or acceptor molecule that could highlight the structural background of the catalytic mechanism. Here, the first crystal structures of α1,6-FucT in complex with its substrate GDP-Fuc and with GDP, which is a byproduct of the enzymatic reaction, are presented. The crystal of the complex with GDP-Fuc was obtained through soaking of native NodZ crystals with the ligand and its structure has been determined at 2.35 Å resolution. The fucose residue is exposed to solvent and is disordered. The enzyme-product complex crystal was obtained by cocrystallization with GDP and an acceptor molecule, penta-N-acetyl-L-glucosamine (penta-NAG). The structure has been determined at 1.98 Å resolution, showing that only the GDP molecule is present in the complex. In both structures the ligands are located in a cleft formed between the two domains of NodZ and extend towards the C-terminal domain, but their conformations differ significantly. The structures revealed that residues in three regions of the C-terminal domain, which are conserved among α1,2-, α1,6- and protein O-fucosyltransferases, are involved in interactions with the sugar-donor molecule. There is also an interaction with the side chain of Tyr45 in the N-terminal domain, which is very unusual for a GT-B-type glycosyltransferase. Only minor conformational changes of the protein backbone are observed upon ligand binding. The only exception is a movement of the loop located between strand βC2 and helix αC3. In addition, there is a shift of the αC3 helix itself upon GDP-Fuc binding.
authors.affiliation: Synchrotron Radiation Research Section, MCL, National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439, USA.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Brzezinski
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Dauter
authors.forename: Mariusz
authors.initials: M
authors.lastname: Jaskolski
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D020369:Bradyrhizobium / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D018360:Crystallography, X-Ray; D005647:Fucosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D006153:Guanosine Diphosphate; D006154:Guanosine Diphosphate Fucose; D005647:Fucosyltransferases; C103941:NodZ protein, bacteria