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Nature. 2020 Mar;579(7799):443-447. doi: 10.1038/s41586-020-2044-z. Epub 2020 Feb 26.

Structure and mechanism of the ER-based glucosyltransferase ALG6.

Author information

1
Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland.
2
Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
3
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA.
4
Institute of Microbiology, ETH Zürich, Zürich, Switzerland.
5
Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland. locher@mol.biol.ethz.ch.

Abstract

In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins1. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates2. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.

PMID:
32103179
DOI:
10.1038/s41586-020-2044-z

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