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Information on EC 2.4.99.18 - dolichyl-diphosphooligosaccharide-protein glycotransferase and Organism(s) Campylobacter jejuni and UniProt Accession Q5HTX9
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2.4.99.18 dolichyl-diphosphooligosaccharide-protein glycotransferaseIUBMB Comments
Occurs in eukaryotes that form a glycoprotein by the transfer of a glucosyl-mannosyl-glucosamine polysaccharide to the side-chain of an L-asparagine residue in the sequence -Asn-Xaa-Ser- or -Asn-Xaa-Thr- (Xaa not Pro) in nascent polypeptide chains. The basic oligosaccharide is the tetradecasaccharide Glc3Man9GlcNAc2 (for diagram {polysacc/Dol14}). However, smaller oligosaccharides derived from it and oligosaccharides with additional monosaccharide units attached may be involved. See ref for a review of N-glycoproteins in eukaryotes. Man3GlcNAc2 seems to be common for all of the oligosaccharides involved with the terminal N-acetylglucosamine linked to the protein L-asparagine. Occurs on the cytosolic face of the endoplasmic reticulum. The dolichol involved normally has 14-21 isoprenoid units with two trans double-bonds at the omega end, and the rest of the double-bonds in cis form.
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Word Map
- 2.4.99.18
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n-glycosylation
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n-linked
- glycans
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lipid-linked
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sequons
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asparagine-linked
- jejuni
- campylobacter
- dolichol
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ribophorins
- otase
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cotranslational
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bloc
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dolichol-linked
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translocon
- glc3man9glcnac2
- heptasaccharide
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s-layer
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underglycosylation
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preassembled
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mannosyltransferases
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glycoengineering
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hypoglycosylation
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hetero-oligomeric
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single-subunit
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translocon-associated
- man9glcnac2
- lari
- drug development
- biotechnology
- medicine
- n-glycoproteins
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glycoproteomics
- pilins
The taxonomic range for the selected organisms is: Campylobacter jejuni
The expected taxonomic range for this enzyme is: Eukaryota, Archaea, Bacteria
The expected taxonomic range for this enzyme is: Eukaryota, Archaea, Bacteria
Reaction Schemes
Synonyms
oligosaccharyltransferase, oligosaccharyl transferase, oligosaccharyltransferase complex, ost3p, wbp1p, ost6p, oligosaccharide transferase, swp1p, tbstt3a, tbstt3b, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
PglB
asparagine N-glycosyltransferase
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-
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dolichyldiphosphooligosaccharide-protein oligosaccharyltransferase
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glycosyltransferase, dolichyldiphosphooligosaccharide-protein
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glycosyltransferase, dolichylpyrophosphodiacetylchitobiose-protein
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oligomannosyltransferase
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oligosaccharide transferase
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oligosaccharyltransferase, dolichyldiphosphoryloligosaccharide-protein
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OST
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PglB
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hexosyl group transfer
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-
-
-
SYSTEMATIC NAME
IUBMB Comments
dolichyl-diphosphooligosaccharide:protein-L-asparagine N-beta-D-oligopolysaccharidotransferase
Occurs in eukaryotes that form a glycoprotein by the transfer of a glucosyl-mannosyl-glucosamine polysaccharide to the side-chain of an L-asparagine residue in the sequence -Asn-Xaa-Ser- or -Asn-Xaa-Thr- (Xaa not Pro) in nascent polypeptide chains. The basic oligosaccharide is the tetradecasaccharide Glc3Man9GlcNAc2 (for diagram {polysacc/Dol14}). However, smaller oligosaccharides derived from it and oligosaccharides with additional monosaccharide units attached may be involved. See ref [2] for a review of N-glycoproteins in eukaryotes. Man3GlcNAc2 seems to be common for all of the oligosaccharides involved with the terminal N-acetylglucosamine linked to the protein L-asparagine. Occurs on the cytosolic face of the endoplasmic reticulum. The dolichol involved normally has 14-21 isoprenoid units with two trans double-bonds at the omega end, and the rest of the double-bonds in cis form.
CAS REGISTRY NUMBER
COMMENTARY
75302-32-8
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
crude lipid-linked oligosaccharide donors from Campylobacter jejuni + carboxytetramethylrhodamine-Ala-Asp-Gln-Asn-Ala-Thr-Tyr-Lys
dolichyl disphosphate + carboxytetramethylrhodamine-Ala-(oligosaccharidyl) Asp-Gln-Asp-Ala-Thr-Tyr
37°C, 50 mM Tris-HCl, pH 7.5
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-
?
dolichyl diphosphooligosaccharide + protein L-asparagine
dolichyl disphosphate + protein with oligosaccharide attached to protein L-asparagine
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-
-
?
dolichyl diphosphooligosaccharide + protein L-asparagine
dolichyl diphosphate + a glycoprotein with the oligosaccharide chain attached by N-glycosyl linkage to protein L-asparagine
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DQNAT is the optimal acceptor substrate. PglB is not capable of utilizing glycosyl donors such as dolichyl-pyrophosphatechitobiose
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-
?
dolichyl diphosphooligosaccharide + [protein]-L-asparagine
dolichyl diphosphate + [protein]-N-oligosaccharidyl-L-asparagine
GalNAc-N,N'-diacetyl-bacillosamine-PP-undecaprenyl + acetyl-DFNAT-(4-nitrophenylalanine)-NH2
?
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?
GalNAc-N,N'-diacetyl-bacillosamine-PP-undecaprenyl + acetyl-DFNVT-(4-nitrophenylalanine)-NH2
?
-
-
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?
GalNAc-N,N'-diacetyl-bacillosamine-PP-undecaprenyl + acetyl-DQNAT-(4-nitrophenylalanine)-NH2
?
-
-
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?
GalNAc-N,N'-diacetyl-bacillosamine-PP-undecaprenyl + acetyl-DVNAS-(4-nitrophenylalanine)-NH2
?
-
-
-
-
?
GalNAc-N,N'-diacetyl-bacillosamine-PP-undecaprenyl + acetyl-DVNAT-(4-nitrophenylalanine)-NH2
?
-
-
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?
GalNAc-N,N'-diacetyl-bacillosamine-PP-undecaprenyl + acetyl-DVNVT-(4-nitrophenylalanine)-NH2
?
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?
GalNAc-N,N'-diacetyl-bacillosamine-PP-undecaprenyl + acetyl-EVNAT-(4-nitrophenylalanine)-NH2
?
-
-
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?
N-acetyl-D-galactosaminyl-alpha-(1->3)-N,N'-diacetyl-alpha-D-bacillosaminyl-diphospho-tritrans,heptacis-undecaprenol + KDFNVSKA
tritrans,heptacis-undecaprenyl diphosphate + Lys-Asp-Phe-N4-[N-acetyl-D-galactosaminyl-alpha-(1->3)-N,N'-diacetyl-beta-D-bacillosaminyl]-Asn-Val-Ser-Lys-Ala
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-
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?
pentaprenyl diphospho-N-acetyl-D-galactosamine + [protein]-L-asparagine
pentaprenyl diphosphate + [protein]-N-(N-acetyl-D-galactosaminyl)-L-asparagine
PglB can utilize shorter polyisoprenol (pentaprenol) as the lipid carrier, albeit with reduced efficiency
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-
?
undecaprenyl diphospho-N-acetyl-D-galactosamine + [protein]-L-asparagine
undecaprenyl diphosphate + [protein]-N-(N-acetyl-D-galactosaminyl)-L-asparagine
PglB is solely responsible for the oligosaccharyltransferase activity. PglB can transfer a monosaccharide, e.g. GalNAc to a peptide acceptor. PglB exhibits relaxed sugar substrate specificity
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?
dolichyl diphosphooligosaccharide + [protein]-L-asparagine
dolichyl diphosphate + [protein]-N-oligosaccharidyl-L-asparagine
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?
dolichyl diphosphooligosaccharide + [protein]-L-asparagine
dolichyl diphosphate + [protein]-N-oligosaccharidyl-L-asparagine
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PglB, the key enzyme of the Campylobacter jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an accept or protein. PglB is the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer
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?
dolichyl diphosphooligosaccharide + [protein]-L-asparagine
dolichyl diphosphate + [protein]-N-oligosaccharidyl-L-asparagine
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PglB has relaxed specificity toward its lipid-linked glycan substrate. It can transfer its endogenous substrate, the heptasaccharide Glc(GalNAc)5Bac of Campylobacter jejuni, as well as different O antigen polysaccharides that are assembled by the rhamnosyltransferase (polymerase)-dependent mechanism on the lipid carrier undecaprenyl diphosphate
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?
dolichyl diphosphooligosaccharide + [protein]-L-asparagine
dolichyl diphosphate + [protein]-N-oligosaccharidyl-L-asparagine
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to investigate the substrate specificity of PglB, the transfer of a set of lipid-linked polysaccharides in Escherichia coli and Salmonella enterica serovar typhimurium is tested. A hexose linked to the C-6 of the monosaccharide at the reducing end does not inhibit the transfer of the O antigen to the acceptor protein. PglB requires an acetamido group at the C-2. A model for the mechanism of PglB involving this functional group is proposed. Eukaryotic and prokaryotic OTases catalyze the transfer of oligosaccharides by a conserved mechanism. Substitution at the C-6 position in the reducing end of the oligosaccharide does not prevent its PglB-mediated transfer to the protein acceptor AcrA. PglB transferrs a polysaccharide that is assembled by a Wzy-protein-independent pathway. An N-acetyl group in position 2 of the sugar directly linked to the undecaprenyl diphosphate carrier is necessary for recognition and/or catalysis
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?
additional information
?
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key enzyme of Campylobacter jejuni N-glycosylation system, transfers O-polysaccharides from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB is the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer
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-
?
additional information
?
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PglB can transfer diverse oligosaccharides and polysaccharides from Escherichia coli and Pseudomonas aeruginosa to proteins, in addition to the Campylobacter jejuni glycan. PglB attaches O7 polysaccharides and Campylobacter jejuni oligosaccharides to the same Asn residue in AcrA
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?
additional information
?
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PglB transfers a wide variety of saccharides
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?
additional information
?
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no activity with acetyl-NVAAT-(para-nitrophenylalanine)-NH2 and acetyl-DVAAT-(para-nitrophenylalanine)-NH2
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
dolichyl diphosphooligosaccharide + protein L-asparagine
dolichyl disphosphate + protein with oligosaccharide attached to protein L-asparagine
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-
-
?
dolichyl diphosphooligosaccharide + [protein]-L-asparagine
dolichyl diphosphate + [protein]-N-oligosaccharidyl-L-asparagine
dolichyl diphosphooligosaccharide + [protein]-L-asparagine
dolichyl diphosphate + [protein]-N-oligosaccharidyl-L-asparagine
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-
-
-
?
dolichyl diphosphooligosaccharide + [protein]-L-asparagine
dolichyl diphosphate + [protein]-N-oligosaccharidyl-L-asparagine
-
PglB, the key enzyme of the Campylobacter jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an accept or protein. PglB is the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer
-
-
?
additional information
?
-
-
key enzyme of Campylobacter jejuni N-glycosylation system, transfers O-polysaccharides from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB is the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer
-
-
?
additional information
?
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-
PglB transfers a wide variety of saccharides
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-
?
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
PglB possesses 11 transmembrane segments and two relatively large periplasmic regions other than the C-terminal domain
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
2.8 A resolution crystal structure of the C-terminal globular domain, hanging drop method
hanging drop crystallization with 10 mM Tris-HCl, pH 8.0, and reservoir solution (0.1 M sodium cacodylate, pH 6.5, 18% polyethylene glycol 8000, 0.2 M calcium acetate, 20°C, cryoprotection with 0.1 M MES, pH 6.5, polyethylene glycol 8000, and 0.2 M calcium acetate)
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D54A
XXD motif mutation, strongly reduced activity compared to wild-type
I571A
MI motif mutation, strongly reduced activity compared to wild-type
N53A
XXD motif mutation, about 40% activity remains compared to wild-type
S52D
XXD motif mutation, strongly reduced activity compared to wild-type
S52E
XXD motif mutation, strongly reduced activity compared to wild-type
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
sonication, centrifugation, supernatant absorbed to glutathione-Sepharose 4B resin, elution, concentration, reductive methylation, gel filtration with Superdex75 column, followed by anion exchange chromatography with a Resource Q column
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
PCR-amplification fused to GST, expressed in Escherichia coli BL21 (DE3)pLysS
the expression plasmid encoding the C-terminal soluble domain (residues 428713) is constructed by inserting a PCR product from genomic DNA (ATCC700819D) into the pGEX-6P-1 vector (GE Healthcare). The GST-fused sPglB protein is expressed by the addition of isopropyl-beta-D-thiogalactopyranoside at 37°C in the Escherichia coli BL21(DE3)pLysS strain. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family reveals three types of oligosaccharyltransferases catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the oligosaccharyltransferases enzymes from eukarya, archaea, and bacteria
PglB is overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures
preparation of a membrane fraction from Escherichia coli in which PglB has been overexpressed
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
medicine
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PglB-mediated transfer of poylsaccharides might be valuable for in vivo production of O-polysaccharides-protein congugates for use as antibacterial vaccines
medicine
-
the development of powerful inhibitors of this enzyme would be valuable in the quest for antibiotics for Campylobacter jejuni-induced gastrointestinal disorders
medicine
-
the relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure is exploited to create novel N-glycan structures containing two distinct Escherichia coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Feldman, M.F.; Wacker, M.; Hernandez, M.; Hitchen, P.G.; Marolda, C.L.; Kowarik, M.; Morris, H.R.; Dell, A.; Valvano, M.A.; Aebi, M.
Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli
Proc. Natl. Acad. Sci. USA
102
3016-3021
2005
Campylobacter jejuni
Glover, K.J.; Weerapana, E.; Numao, S.; Imperiali, B.
Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni
Chem. Biol.
12
1311-1315
2005
Campylobacter jejuni
Chen, M.M.; Glover, K.J.; Imperiali, B.
From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni
Biochemistry
46
5579-5585
2007
Campylobacter jejuni
Li, L.; Woodward, R.; Ding, Y.; Liu, X.W.; Yi, W.; Bhatt, V.S.; Chen, M.; Zhang, L.W.; Wang, P.G.
Overexpression and topology of bacterial oligosaccharyltransferase PglB
Biochem. Biophys. Res. Commun.
394
1069-1074
2010
Campylobacter jejuni (Q9S4V7), Campylobacter jejuni
Maita, N.; Nyirenda, J.; Igura, M.; Kamishikiryo, J.; Kohda, D.
Comparative structural biology of eubacterial and archaeal oligosaccharyltransferases
J. Biol. Chem.
285
4941-4950
2010
Campylobacter jejuni (Q5HTX9), Campylobacter jejuni
Wacker, M.; Feldman, M.F.; Callewaert, N.; Kowarik, M.; Clarke, B.R.; Pohl, N.L.; Hernandez, M.; Vines, E.D.; Valvano, M.A.; Whitfield, C.; Aebi, M.
Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems
Proc. Natl. Acad. Sci. USA
103
7088-7093
2006
Campylobacter jejuni
EXTERNAL LINKS (specific for EC-Number 2.4.99.18)
Transporter Classification Database (TCDB): 9.B.142.3.17, 9.B.142.3.14, 9.B.142.3.4, 9.B.142.3.5, 9.B.142.3.3
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