Structural and mechanistic studies of the N-glycosylation machinery: From lipid-linked oligosaccharide biosynthesis to glycan transfer.

N-linked protein glycosylation is a post-translational modification that exists in all domains of life. It involves two consecutive steps: (1) biosynthesis of a lipid-linked oligosaccharide (LLO), and (2) glycan transfer from the LLO to asparagine residues in secretory proteins, which is catalyzed by the integral membrane enzyme oligosaccharyltransferase (OST). In the last decade, structural and functional studies of the N-glycosylation machinery have increased our mechanistic understanding of the pathway. The structures of bacterial and eukaryotic glycosyltransferases involved in LLO elongation provided insight into the mechanism of LLO biosynthesis, whereas structures of OST enzymes revealed the molecular basis of sequon recognition and catalysis. In this review, we will discuss approaches used and insight obtained from these studies with a special emphasis on the design and preparation of substrate analogs.