Asparagine-linked glycosylation is a common posttranslational modification of diverse secretory and membrane proteins in eukaryotes, where it is catalyzed by the multiprotein complex oligosaccharyltransferase. The functions of the protein subunits of oligoasccharyltransferase, apart from the catalytic Stt3p, are ill defined. Here we describe functional and structural investigations of the Ost3/6p components of the yeast enzyme. Genetic, biochemical and structural analyses of the lumenal domain of Ost6p revealed oxidoreductase activity mediated by a thioredoxin-like fold with a distinctive active-site loop that changed conformation with redox state. We found that mutation of the active-site cysteine residues of Ost6p and its paralogue Ost3p affected the glycosylation efficiency of a subset of glycosylation sites. Our results show that eukaryotic oligosaccharyltransferase is a multifunctional enzyme that acts at the crossroads of protein modification and protein folding.crystal structure ͉ mass spectrometry ͉ N-glycosylation ͉ protein folding ͉ thioredoxin-like protein

N-linked glycosylation is one of the most abundant modifications of proteins in eukaryotic organisms. In the central reaction of the pathway, oligosaccharyltransferase (OST), a multimeric complex located at the membrane of the endoplasmic reticulum, transfers a preassembled oligosaccharide to selected asparagine residues within the consensus sequence asparagine-X-serine/threonine. Due to the high substrate specificity of OST, alterations in the biosynthesis of the oligosaccharide substrate result in the hypoglycosylation of many different proteins and a multitude of symptoms observed in the family of congenital disorders of glycosylation (CDG) type I. This review covers our knowledge of human OST and describes enzyme composition. The Stt3 subunit of OST harbors the catalytic center of the enzyme, but the function of the other, highly conserved, subunits are less well defined. Some components seem to be involved in the recognition and utilization of glycosylation sites in specific glycoproteins. Indeed, mutations in the subunit paralogs N33/Tusc3 and IAP do not yield the pleiotropic phenotypes typical for CDG type I but specifically result in nonsyndromic mental retardation, suggesting that the oxidoreductase activity of these subunits is required for glycosylation of a subset of proteins essential for brain development.

The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis highlights the need to discover new antitubercular agents. Here we describe the synthesis and characterization of a new series of thienopyrimidine (TP) compounds that kill both replicating and non-replicating M. tuberculosis. The strategy to determine the mechanism of action of these TP derivatives was to generate resistant mutants to the most effective compound TP053 and to isolate the genetic mutation responsible for this phenotype. The only non-synonymous mutation found was a g83c transition in the Rv2466c gene, resulting in the replacement of tryptophan 28 by a serine. The Rv2466c overexpression increased the sensitivity of M. tuberculosis wild-type and resistant mutant strains to TP053, indicating that TP053 is a prodrug activated by Rv2466c. Biochemical studies performed with purified Rv2466c demonstrated that only the reduced form of Rv2466c can activate TP053. The 1.7 Å resolution crystal structure of the reduced form of Rv2466c, a protein whose expression is transcriptionally regulated during the oxidative stress response, revealed a unique homodimer in which a β-strand is swapped between the thioredoxin domains of each subunit. A pronounced groove harboring the unusual active-site motif CPWC might account for the uncommon reactivity profile of the protein. The mutation of Trp28Ser clearly predicts structural defects in the thioredoxin fold, including the destabilization of the dimerization core and the CPWC motif, likely impairing the activity of Rv2466c against TP053. Altogether our experimental data provide insights into the molecular mechanism underlying the anti-mycobacterial activity of TP-based compounds, paving the way for future drug development programmes.

N-linked glycosylation of proteins in the endoplasmic reticulum (ER) is essential in eukaryotes and catalyzed by oligosaccharyl transferase (OST). Human OST is a hetero-oligomer of seven subunits. The subunit N33/Tusc3 is a tumor suppressor candidate, and defects in the subunit N33/Tusc3 are linked with nonsyndromic mental retardation. Here, we show that N33/Tusc3 possesses a membrane-anchored N-terminal thioredoxin domain located in the ER lumen that may form transient mixed disulfide complexes with OST substrates. X-ray structures of complexes between N33/Tusc3 and two different peptides as model substrates reveal a defined peptide-binding groove adjacent to the active site that can accommodate peptides in opposite orientations. Structural and biochemical data show that N33/Tusc3 prefers peptides bearing a hydrophobic residue two residues away from the cysteine forming the mixed disulfide with N33/Tusc3. Our results support a model in which N33/Tusc3 increases glycosylation efficiency for a subset of human glycoproteins by slowing glycoprotein folding.

Background: ␣-Proteobacteria, extant relatives of mitochondria, are model organisms for studying assembly of bacterial and mitochondrial metalloenzymes. Results: Periplasmic thioredoxin TlpA is a specific reductant for copper chaperone ScoI and cytochrome oxidase subunit II (CoxB). Conclusion: Cysteines in the copper-binding sites of ScoI and CoxB must be reduced prior to metallation. Significance: Structures of TlpA-ScoI and TlpA-CoxB intermediates reveal mechanistic details of the reduction process.