Comparative Structural Biology of Eubacterial and Archaeal Oligosaccharyltransferases

Abstract

Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. In the bacterium Campylobacter jejuni, a single-subunit membrane protein, PglB, catalyzes Nglycosylation. We report the 2.8 Å resolution crystal structure of the C-terminal globular domain of PglB and its comparison with the previously determined structure from the archaeon Pyrococcus AglB. The two distantly related oligosaccharyltransferases share unexpected structural similarity beyond that expected from the sequence comparison. The common architecture of the putative catalytic sites revealed a new catalytic motif in PglB. Site-directed mutagenesis analyses confirmed the contribution of this motif to the catalytic function. Bacterial PglB and archaeal AglB constitute a protein family of the catalytic subunit of OST along with STT3 from eukaryotes. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family revealed three types of OST catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the OST enzymes from Eukarya, Archaea, and Bacteria.Protein N-glycosylation is an important posttranslational modification that occurs in all domains of life (1). The enzyme that creates the oligosaccharide-asparagine linkage is oligosaccharyltransferase (OST).5 OST catalyzes the en bloc transfer of a preassembled oligosaccharide from a lipid carrier to asparagine residues in the glycosylation consensus (Asn-X-Thr/Ser, where X represents any amino acid except for Pro) of polypeptide chains (2-4). OST is a multisubunit membrane protein complex in higher eukaryotes. Yeast (Saccharomyces cerevisiae) OST consists of eight different subunits: Ost1p, Ost2p, Ost3p/ Ost6p, Ost4p, Wbp1, Swp1, Stt3p, and Ost5p (5), where Ost3p and Ost6p are paralogs that are present in two distinct OST isoforms (6). The cryoelectron microscopy structure of the digitonin-solubilized OST complex from yeast provided the relative arrangement of Ost1p, Wbp1, and Stt3p on the lumenal side of the complex (7,8).Stt3p is the catalytic subunit of the yeast OST enzyme (9). The vertebrate, insect, and plant equivalents are the two paralog proteins, STT3A and STT3B, which define distinct OST isoforms (10, 11). In lower eukaryotes, such as trypanosomatids, OST is a single-polypeptide membrane protein (3), and these single-subunit OST proteins consist of STT3 (staurosporine and temperature sensitivity 3) alone. In fact, the STT3s from Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major can each function as an OST enzyme when transferred into stt3-deficient yeast cells (12)(13)(14)(15). The prokaryotic OST is also a single-polypeptide protein. The STT3 homologs, PglB (protein glycosylation B) and AglB (archaeal glycosylation B), comprise the bacterial OST and the archaeal OST,. Multiple STT3/PglB/AglB paralogs also exist in some single-subunit OSTs. The trypanosomatids L. major and T. brucei contain four and three STT3 par...

Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation

“…8). This view is justified by the higher homology of the amino acid sequences of the crenarchaeal AglB enzymes to the eukaryotic STT3 counterparts than to those of the euryarchaeal AglBs (5). The N-glycosylation in the euryarchaeal species is dispensable (36,37), but the disruption of the N-glycosylation system is reportedly lethal in the crenarchaeal species (38), as in Eukaryotes.…”

Substrate and Metal Ion Promiscuity in Mannosylglycerate Synthase

“…Surprisingly, given the sequence diversity represented across GT families, predominantly only two structural folds are adopted by nucleotide sugar-dependent GTs; termed GT-A and GT-B (6,8). A third fold class, GT-C, has recently been proposed for the transmembrane-spanning GT family 66, which utilize lipid phosphate-activated sugars as the donor substrate (9,10).…”

Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation

“…We previously determined the crystal structures of the C-terminal globular domains of the three paralogous AglB proteins from Archaeoglobus fulgidus (21)(22)(23), as well as two other AglB proteins and one PglB protein (14,22,24). The structural comparison revealed a common structural unit that contains the Ser/ Thr pocket, and other structural units specific to each protein.…”