Structure and mechanism of the ER-based glucosyltransferase ALG6

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biocatalysis
  • Catalytic Domain
  • Conserved Sequence
  • Cryoelectron Microscopy*
  • Dolichol Monophosphate Mannose / metabolism
  • Dolichol Phosphates / metabolism
  • Endoplasmic Reticulum / enzymology*
  • Glucose / analogs & derivatives
  • Glucose / metabolism
  • Glycosyltransferases / deficiency
  • Glycosyltransferases / genetics*
  • Glycosyltransferases / metabolism*
  • In Vitro Techniques
  • Lipids
  • Membrane Proteins / deficiency
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism*
  • Models, Molecular
  • Mutation
  • Polyisoprenyl Phosphate Monosaccharides / chemistry
  • Polyisoprenyl Phosphate Monosaccharides / metabolism
  • Protein Binding
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Substrate Specificity


  • Dolichol Phosphates
  • Lipids
  • Membrane Proteins
  • Polyisoprenyl Phosphate Monosaccharides
  • Saccharomyces cerevisiae Proteins
  • dolichol monophosphate
  • Dolichol Monophosphate Mannose
  • dolichol-D-glucosylmonophosphate
  • Glycosyltransferases
  • ALG6 protein, S cerevisiae
  • Glucose