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Structural, mutagenic and in silico studies of xyloglucan fucosylation in Arabidopsis thaliana suggest a water‐mediated mechanism
Author(s) -
Urbanowicz Breeanna R.,
Bharadwaj Vivek S.,
Alahuhta Markus,
Peña Maria J.,
Lunin Vladimir V.,
Bomble Yannick J.,
Wang Shuo,
Yang JeongYeh,
Tuomivaara Sami T.,
Himmel Michael E.,
Moremen Kelley W.,
York William S.,
Crowley Michael F.
Publication year - 2017
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13628
Subject(s) - fucosylation , fucosyltransferase , xyloglucan , mutagenesis , transferase , arabidopsis , glycosyltransferase , arabidopsis thaliana , biochemistry , mutant , chemistry , biosynthesis , enzyme , stereochemistry , fucose , gene , galactose
Summary 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 ( At FUT1) 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 At FUT1 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 At FUT1 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.