The resistance of glycosyltransferase (GT) enzymes to structural characterization has clouded our understanding of the mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis. The enzyme fucosyltransferase-1 (AtFUT1) catalyzes the regiospecific transfer of terminal 1,2-fucosyl residues to xyloglucan side chains - the final step of hemicellulose biosynthesis - in Arabidopsis thaliana. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving molecular simulations enabled by protein expression, X-ray crystallography. 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. Molecular dynamics simulations of the enzyme substrate complex reveals the presence of aptly positioned water molecules at the active site. Quantum mechanical calculations of a reduced active site suggest a water-mediated fucosylation mechanism, which is cooroborated by mutagenesis experiments.

Unravelling hemicellulose bio-synthesis using molecular simulations

254th National Meeting (2017)

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