title: Expression, purification and biochemical characterization of AtFUT1, a xyloglucan-specific fucosyltransferase from Arabidopsis thaliana.
abstract: 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-β-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.
authors: Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France.
authors: Félix
authors: F
authors: Cicéron
authors: Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France.
authors: Joana
authors: J
authors: Rocha
authors: Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France.
authors: Sumaira
authors: S
authors: Kousar
authors: Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France.
authors: Sara Fasmer
authors: SF
authors: Hansen
authors: Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France.
authors: Valérie
authors: V
authors: Chazalet
authors: Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France.
authors: Emilie
authors: E
authors: Gillon
authors: Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France.
authors: Christelle
authors: C
authors: Breton
authors: Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France. Electronic address: lerouxel@cermav.cnrs.fr.
authors: Olivier
authors: O
authors: Lerouxel
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002850:Chromatography, Gel; D005647:Fucosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005936:Glucans / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D009036:Moths / Q000166:cytology; D011485:Protein Binding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D005936:Glucans; D011994:Recombinant Proteins; D012996:Solutions; D014990:Xylans; D006154:Guanosine Diphosphate Fucose; C029353:xyloglucan; D005647:Fucosyltransferases; C070983:xyloglucan 2-fucosyltransferase
keywords: AtFUT1; Fucosyltransferase; ITC; Insect cell expression; Xyloglucan