Journal
PLANT JOURNAL
Volume 91, Issue 6, Pages 931-949Publisher
WILEY
DOI: 10.1111/tpj.13628
Keywords
Arabidopsis thaliana; fucosylation; hemicellulose synthesis; fucosyltransferase; reaction mechanism
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Funding
- BioEnergy Science Center (BESC), a US Department of Energy Bioenergy Research Center - Office of Biological and Environmental Research in the US Department of Energy Office of Science
- National Institutes of Health (NIH) [P41GM103390, P01 GM107012]
- DOE Office of EERE [DE-AC36-08GO28308]
- Extreme Science and Engineering Discovery Environment (XSEDE)
- National Science Foundation [ACI-1053575]
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The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely because of 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 the 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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