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

Cicéron, Félix; Rocha, Joana; Kousar, Sumaira; Hansen, Sara Fasmer; Chazalet, V.; Gillon, Émilie; Breton, C.; Lerouxel, Olivier

doi:10.1016/j.biochi.2016.08.012

Cited by 12 publications

(22 citation statements)

References 34 publications

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“…The structures of peaks a and b were deduced to be XFLG or XLFG for peak a and XJLG or XLJG for peak b, where J denotes l ‐Galα1,2‐ d ‐Galβ1,2Xylα1,6Glc respectively. These results support the hypothesis that the AtFUT1 exhibits l ‐galactosyltransferase activity on XLLG oligosaccharide when GDP‐ l ‐Gal is used as a substrate, in addition to the reported l ‐fucosyltransferase activity .…”

Section: Resultssupporting

confidence: 88%

“…This may also be the reason why only mono-L-galactosylated XLLGXLLG-PA was detected, and the nonreducing end-portion of the XLLG unit in XLLGXLLG-PA was likely L-galactosylated. Moreover, AtFUT1 showed the faster initial velocity of L-galactosylation toward the longer XLLGXLLG-PA oligosaccharides than to the shorter, nonlabeled XLLG oligosaccharide, as is also the case with L-fucosylation [15,16], indicating that AtFUT1 recognizes not only the single XLLG unit but also at least 2 repeated XLLG units. The tertiary structure of the acceptor-binding site recognizing the reducing-end Glc residue in XLLG oligosaccharide is not fully available.…”

Section: Discussionmentioning

confidence: 78%

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

Ohashi

Misaki

et al. 2018

FEBS Letters

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l‐Galactose (l‐Gal) is one of the components of plant cell wall polysaccharides. In the GDP‐l‐fucose‐deficient Arabidopsis thaliana mutant mur1, l‐fucose (l‐Fuc) residues in xyloglucan are substituted by l‐Gal residues. l‐Gal only differs from l‐Fuc by the presence of an oxygen at C‐6. Thus, we hypothesized that the A. thaliana xyloglucan α1,2‐l‐fucosyltransferase (AtFUT1) is also responsible for the l‐galactosyl transfer to d‐galactose residues in xyloglucan. In this study, we heterologously produced AtFUT1 in fission yeast and carried out an in vitro assay for the activities of AtFUT1 on GDP‐l‐Gal and xyloglucan oligosaccharide. We show that the recombinant AtFUT1 catalyzes l‐Gal transfer to xyloglucan oligosaccharides although the initial velocity of l‐Gal transfer is 3.1 times lower than that of l‐Fuc transfer.

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Section: Resultssupporting

confidence: 88%

Section: Discussionmentioning

confidence: 78%

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

Ohashi

Misaki

et al. 2018

FEBS Letters

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“…Furthermore, as GTs can also transfer sugar (from the nucleotide donor) to water in the absence of an acceptor, albeit at a low but significant rate, the enzymatic properties of At FUT1 were evaluated by measuring both the nucleotide sugar hydrolase and fucosyltransferase activities for the WT enzyme and several mutant variants by measuring the production of GDP from GDP‐Fuc (GDP‐Glo TM ) in the presence and absence of XXLG (Figure ). The kinetic parameters of the wild‐type (WT) enzyme with GDP‐Fuc (Figure S5; Table ) are consistent with those previously reported for the FUT1 ortholog from Pisum sativum (PsFT1) and for HisΔ68‐ At FUT1 (Faik et al ., ; Cicéron et al ., ). Interestingly, although WT At FUT1 is able to hydrolyze GDP‐fucose, our data indicate that the rate of hydrolysis is at least 80‐fold lower than fucosyltransfer (Figure ).…”

Section: Resultsmentioning

confidence: 97%

“…The yields that we were able to achieve using HEK‐based expression are exceedingly higher than the recombinant expression of HisΔ68‐ At FUT1 carried out by Cicéron et al . () 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 ., ).…”

Section: Resultsmentioning

confidence: 99%

“…() 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.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely due to the resistance of glycosyltransferase (GT) enzymes to structural characterization. In Arabidopsis thaliana, a glycosyl transferase family 37 (GT37) fucosyltransferase-1 (AtFUT1) catalyzes the regiospecific transfer of terminal 1,2-fucosyl residues to xyloglucan side chains – a key step in the biosynthesis of fucosylated sidechains of galactoxyloglucan. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving protein expression, X-ray crystallography, mutagenesis experiments and molecular simulations. Mammalian cell culture expressions enable sufficient production of the enzyme for X-ray crystallography, which reveals the structural architecture of AtFUT1 in complex with bound donor and acceptor substrate analogs. The lack of an appropriately positioned active site residue as a catalytic base leads us to propose an atypical water-mediated fucosylation mechanism facilitated by an H-bonded network, which is corroborated by mutagenesis experiments as well as detailed atomistic simulations.

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Analysis of Carbohydrates and Glycoconjugates by Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry: An Update for 2015–2016

Harvey

2021

Mass Spectrometry Reviews

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This review is the ninth update of the original article published in 1999 on the application of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2016. Also included are papers that describe methods appropriate to analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation and arrays. The second part of the review is devoted to applications to various structural types such as oligo‐ and poly‐saccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions and applications to chemical synthesis. The reported work shows increasing use of combined new techniques such as ion mobility and the enormous impact that MALDI imaging is having. MALDI, although invented over 30 years ago is still an ideal technique for carbohydrate analysis and advancements in the technique and range of applications show no sign of deminishing. © 2020 Wiley Periodicals, Inc.

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

Cited by 12 publications

References 34 publications

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

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

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

Analysis of Carbohydrates and Glycoconjugates by Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry: An Update for 2015–2016

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