Literature summary for 2.4.99.18 extracted from

Matsumoto, S.; Igura, M.; Nyirenda, J.; Matsumoto, M.; Yuzawa, S.; Noda, N.; Inagaki, F.; Kohda, D.
Crystal structure of the C-terminal globular domain of oligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 A resolution (2012), Biochemistry, 51, 4157-4166.

Search for more literature for 2.4.99.18

Cloned(Commentary)

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 (Commentary)

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

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

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

Metals/Ions	Comment	Organism	Structure
Mn2+	activates	Archaeoglobus fulgidus

Natural Substrates/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Archaeoglobus fulgidus	O29918	NBRC 100126, three AglB paralogues	-
Archaeoglobus fulgidus DSM 4306	O29918	NBRC 100126, three AglB paralogues	-

Purification (Commentary)

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 and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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

Synonyms	Comment	Organism
oligosaccharyltransferase	-	Archaeoglobus fulgidus
OST	-	Archaeoglobus fulgidus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
60	-	assay at	Archaeoglobus fulgidus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Archaeoglobus fulgidus

General Information

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