Comparative Structural Biology of Eubacterial and Archaeal Oligosaccharyltransferases

Nobuo Maita,James Nyirenda,Jun Kamishikiryo,Mayumi Igura,Daisuke Kohda

doi:10.1074/jbc.m109.081752

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 N-glycosylation. We report the 2.8 A 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.

Highlights

We found a pair of Asp and Lys residues spaced three residues apart (DXXK, where X can be any residue) that is conserved in yeast Stt3p, the two Pyrococcus AglB paralogs, and Campylobacter PglB and named it the “DK” motif
This alignment seems valid between eukaryotic STT3 and archaeal AglB, because in vivo mutational studies indicated that the Asp and Lys residues in the motif were catalytically important in yeast Stt3p and in L. major STT3–1 [14, 31]
C-terminal globular domain was expressed as a fusion protein in

Summary

Introduction

The C-terminal half of the primary sequence forms a globular domain bearing a well conserved five-residue motif, WWDYG. A meaningful multiple sequence alignment of STT3, PglB, and AglB across the three domains of life is virtually impossible, except for the vicinity of the WWDYG motif, due to the very low sequence homology among them. This alignment seems valid between eukaryotic STT3 and archaeal AglB, because in vivo mutational studies indicated that the Asp and Lys residues in the motif were catalytically important in yeast Stt3p and in L. major STT3–1 [14, 31].

Results

Conclusion