Cloned (Comment) | Organism |
---|---|
cloning of DNA encoding the full-length AfAglB-S1 gene, expression of N-terminally His6-tagged wild-type and selenomethionine-labeled AglB in Escherichia coli strains BL21(DE3) and C43(DE3), respectively | Archaeoglobus fulgidus |
Crystallization (Comment) | Organism |
---|---|
C-terminal globular domain of the of the smallest AglB, purified recombinant wild-type and selenomethionine-labeled AgBs, sitting and hanging drop vapor diffusion methods, crystals grow from a hanging drop with a 1:1 volume ratio of the protein stock solution containing 20 mg/mL protein in 20 mM Tris buffer, pH 8.0, and the reservoir solution containing 0.1 M MES buffer, pH 6.5, and 12.5% PEG 3350, at 20°C, soaking of crystals in reservoir solution with 20% ethylene glycol added for cryoprotection, X-ray diffraction structure determination and analysis at 1.75-1.94 A resolution | Archaeoglobus fulgidus |
Protein Variants | Comment | Organism |
---|---|---|
D520A | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli | Archaeoglobus fulgidus |
D520N | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
D526A | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli | Archaeoglobus fulgidus |
E521A | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli | Archaeoglobus fulgidus |
E521Q | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
I522A | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
I535A | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli | Archaeoglobus fulgidus |
K531A | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows similar OST activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
K531D | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
K531E | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
K531R | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
M524A | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli | Archaeoglobus fulgidus |
N528A | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
T518A | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant cannot be expressed in Escherichia coli | Archaeoglobus fulgidus |
T523A | site-directed mutagensis of the AglB-S1 subunit of OST, the mutant shows reduced OST activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mn2+ | activates | Archaeoglobus fulgidus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
dolichyl diphosphooligosaccharide + [protein]-L-asparagine | Archaeoglobus fulgidus | - |
dolichyl diphosphate + [protein]-L-asparagine-N-oligosaccharide | a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine | ? | |
dolichyl diphosphooligosaccharide + [protein]-L-asparagine | Archaeoglobus fulgidus DSM 4306 | - |
dolichyl diphosphate + [protein]-L-asparagine-N-oligosaccharide | a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine | ? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Archaeoglobus fulgidus | O29918 | NBRC 100126, three AglB paralogues | - |
Archaeoglobus fulgidus DSM 4306 | O29918 | NBRC 100126, three AglB paralogues | - |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His6-tagged wild-type and selenomethionine-labeled AglB from Escherichia coli strain BL21 by nickel affinity chromatography and gel filtration, followed by anion exchange chromatography | Archaeoglobus fulgidus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
dolichyl diphosphooligosaccharide + [protein]-L-asparagine | - |
Archaeoglobus fulgidus | dolichyl diphosphate + [protein]-L-asparagine-N-oligosaccharide | a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine | ? | |
dolichyl diphosphooligosaccharide + [protein]-L-asparagine | - |
Archaeoglobus fulgidus DSM 4306 | dolichyl diphosphate + [protein]-L-asparagine-N-oligosaccharide | a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine | ? | |
dolichyl diphosphooligosaccharide + [protein]-L-asparagine | i.e. substrate peptide LLO prepared from Pyrococcus furiosus cells and a substrate peptide, TAMRAAla-Pro-Tyr-Asn-Val-Thr-Lys-Arg, which is optimized by peptide library experiments | Archaeoglobus fulgidus | dolichyl diphosphate + [protein]-L-asparagine-oligosaccharide | a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine, product analysis by mass spectrometry | ? | |
dolichyl diphosphooligosaccharide + [protein]-L-asparagine | i.e. substrate peptide LLO prepared from Pyrococcus furiosus cells and a substrate peptide, TAMRAAla-Pro-Tyr-Asn-Val-Thr-Lys-Arg, which is optimized by peptide library experiments | Archaeoglobus fulgidus DSM 4306 | dolichyl diphosphate + [protein]-L-asparagine-oligosaccharide | a glycoprotein with the oligosaccharide chain attached by N-beta-D-glycosyl linkage to a protein L-asparagine, product analysis by mass spectrometry | ? | |
additional information | a set of peptides, TAMRA-Gly-X-X-X-Val-Thr-NH2, where the X-X-X sequence is varied for testing the N-glycosylation dependency, is validated as substrates | Archaeoglobus fulgidus | ? | - |
? | |
additional information | a set of peptides, TAMRA-Gly-X-X-X-Val-Thr-NH2, where the X-X-X sequence is varied for testing the N-glycosylation dependency, is validated as substrates | Archaeoglobus fulgidus DSM 4306 | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | Archaeoglobus AglB lacks a beta-barrel-like structure found in other AglBs and PglBs,but AglB contains a kinked helix bearing a conserved motif, called DK/MI motif, it is a variant type of the DK motif with the insertion, this particular type of the DK motif contributes to the activity. Structure comparisons, overview | Archaeoglobus fulgidus |
Synonyms | Comment | Organism |
---|---|---|
oligosaccharyltransferase | - |
Archaeoglobus fulgidus |
OST | - |
Archaeoglobus fulgidus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
60 | - |
assay at | Archaeoglobus fulgidus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Archaeoglobus fulgidus |
General Information | Comment | Organism |
---|---|---|
evolution | the catalytic subunit of OST is called STT3 in eukaryotes, AglB in archaea, and PglB in eubacteria. Archaeoglobus f ulgidus encodes three AglB paralogues, two of them are the shortest AglBs across all domains of life. | Archaeoglobus fulgidus |
physiological function | oligosaccharyltransferase transfers glycan to asparagine in the N-glycosylation sequon, the lysine and isoleucine residues in the DK/MI motif participate in the Ser/Thr recognition in the sequon | Archaeoglobus fulgidus |