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

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. Here, 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.
Authors:
 [1] ; ORCiD logo [2] ;  [2] ;  [1] ;  [2] ;  [2] ;  [1] ;  [1] ;  [3] ;  [2] ;  [1] ;  [1] ;  [2]
  1. Univ. of Georgia, Athens, GA (United States)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  3. Univ. of Georgia, Athens, GA (United States); Univ. of California San Francisco, San Francisco, CA (United States)
Publication Date:
Report Number(s):
NREL/JA-2700-67766
Journal ID: ISSN 0960-7412
Grant/Contract Number:
AC36-08GO28308
Type:
Accepted Manuscript
Journal Name:
The Plant Journal
Additional Journal Information:
Journal Volume: 91; Journal Issue: 6; Journal ID: ISSN 0960-7412
Publisher:
Society for Experimental Biology
Research Org:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); USDOE Office of Energy Efficiency and Renewable Energy (EERE)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; Arabidopsis thaliana; fucosylation; hemicellulose synthesis; fucosyltransferase; reaction mechanism
OSTI Identifier:
1389734
Alternate Identifier(s):
OSTI ID: 1377643