Glycobiology and Extracellular Matrices
Structural basis of substrate recognition and catalysis by fucosyltransferase 8

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Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80–2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

glycobiology
fucosyltransferase
glycosyltransferase
substrate specificity
glycosylation
N-linked glycosylation
structural biology
enzyme mechanism
core fucose
enzyme evolution
fucosyltransferase 8 (FUT8)
protein maturation

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This work was supported by The Walter and Eliza Hall Institute of Medical Research, National Health and Medical Research Council of Australia Project Grant GNT1139549, the Australian Cancer Research Fund, and a Victorian State Government Operational Infrastructure support grant. The authors declare that they have no conflicts of interest with the contents of this article.

This article contains Figs. S1–S4 and Table S1.

The atomic coordinates and structure factors (codes 6VLD, 6VLE, 6VLF, and 6VLG) have been deposited in the Protein Data Bank (http://wwpdb.org/).

1

Both authors contributed equally to this work.