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Literature summary extracted from

  • Maita, N.; Nyirenda, J.; Igura, M.; Kamishikiryo, J.; Kohda, D.
    Comparative structural biology of eubacterial and archaeal oligosaccharyltransferases (2010), J. Biol. Chem., 285, 4941-4950.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.99.18 PCR-amplification fused to GST, expressed in Escherichia coli BL21 (DE3)pLysS Campylobacter jejuni
2.4.99.18 the expression plasmid encoding the C-terminal soluble domain (residues 428–713) 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 Campylobacter jejuni
2.4.99.19 gene pglB, expression of the GST-tagged C-terminal globular domain of PglB in Escherichia coli strain BL21(DE3)pLysS, expression of His-tagged full-length PglB in Escherichia coli strain BL21(DE3) Campylobacter jejuni

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.4.99.18 2.8 A resolution crystal structure of the C-terminal globular domain, hanging drop method Campylobacter jejuni
2.4.99.18 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) Campylobacter jejuni
2.4.99.19 purified detagged and methylated wild-type and selenomethionine-labeled C-terminal globular domain of PglB, hanging drop vapour diffusion method, 500 nl of 10 mg/ml protein in 10 mM Tris-HCl, pH 8.0, is mixed in an 1:1 ratio with reservoir solution containing 0.1 M sodium cacodylate, pH 6.5, 18% PEG 8000, 0.2 M calcium acetate, 20°C, X-ray diffraction structure determination and analysis at 2.8 A resolution Campylobacter jejuni

Protein Variants

EC Number Protein Variants Comment Organism
2.4.99.18 D54A XXD motif mutation, strongly reduced activity compared to wild-type Campylobacter jejuni
2.4.99.18 I571A MI motif mutation, strongly reduced activity compared to wild-type Campylobacter jejuni
2.4.99.18 L570A MI motif mutation, similar activity as wild-type Campylobacter jejuni
2.4.99.18 M568A MI motif mutation, similar activity as wild-type Campylobacter jejuni
2.4.99.18 N53A XXD motif mutation, about 40% activity remains compared to wild-type Campylobacter jejuni
2.4.99.18 S52D XXD motif mutation, strongly reduced activity compared to wild-type Campylobacter jejuni
2.4.99.18 S52E XXD motif mutation, strongly reduced activity compared to wild-type Campylobacter jejuni
2.4.99.18 S569A MI motif mutation, similar activity as wild-type Campylobacter jejuni

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
2.4.99.18 membrane
-
 Campylobacter jejuni 16020
-
2.4.99.19 membrane
-
 Campylobacter jejuni 16020
-

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.4.99.18 dolichyl diphosphooligosaccharide + protein L-asparagine Campylobacter jejuni
-
 dolichyl disphosphate + protein with oligosaccharide attached to protein L-asparagine
-
 ?
2.4.99.18 dolichyl diphosphooligosaccharide + protein L-asparagine Campylobacter jejuni RM1221
-
 dolichyl disphosphate + protein with oligosaccharide attached to protein L-asparagine
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.4.99.18 Campylobacter jejuni Q5HTX9
-
-
2.4.99.19 Campylobacter jejuni Q5HTX9 gene pglB
-
2.4.99.19 Campylobacter jejuni RM1221 Q5HTX9 gene pglB
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.99.18 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 Campylobacter jejuni
2.4.99.19 recombinant His-tagged full-length PglB from Escherichia coli strain BL21(DE3) in the membrane fraction by ultracentrifugation, recombinant GST-tagged wild-type and selenomethionine-labeled C-terminal globular domain of PglB from Escherichia coli strain BL21(DE3)pLysS by glutathione affinity chromatography and cleavage of the tag by 3C protease, followed by to reductive methylation of the lysine residues, gel filtration, and anion exchange chromatography Campylobacter jejuni

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.99.18 crude lipid-linked oligosaccharide donors from Campylobacter jejuni + carboxytetramethylrhodamine-Ala-Asp-Gln-Asn-Ala-Thr-Tyr-Lys 37°C, 50 mM Tris-HCl, pH 7.5 Campylobacter jejuni dolichyl disphosphate + carboxytetramethylrhodamine-Ala-(oligosaccharidyl) Asp-Gln-Asp-Ala-Thr-Tyr
-
 ?
2.4.99.18 crude lipid-linked oligosaccharide donors from Campylobacter jejuni + carboxytetramethylrhodamine-Ala-Asp-Gln-Asn-Ala-Thr-Tyr-Lys 37°C, 50 mM Tris-HCl, pH 7.5 Campylobacter jejuni RM1221 dolichyl disphosphate + carboxytetramethylrhodamine-Ala-(oligosaccharidyl) Asp-Gln-Asp-Ala-Thr-Tyr
-
 ?
2.4.99.18 dolichyl diphosphooligosaccharide + protein L-asparagine
-
 Campylobacter jejuni dolichyl disphosphate + protein with oligosaccharide attached to protein L-asparagine
-
 ?
2.4.99.18 dolichyl diphosphooligosaccharide + protein L-asparagine
-
 Campylobacter jejuni RM1221 dolichyl disphosphate + protein with oligosaccharide attached to protein L-asparagine
-
 ?

Subunits

EC Number Subunits Comment Organism
2.4.99.19 monomer
-
 Campylobacter jejuni
2.4.99.19 More C-terminal globular domain of PglB compared with the structure from the archaeon Pyrococcus AglB, overview Campylobacter jejuni

Synonyms

EC Number Synonyms Comment Organism
2.4.99.18 oligosaccharyltransferase
-
 Campylobacter jejuni
2.4.99.18 OST
-
 Campylobacter jejuni
2.4.99.18 PglB
-
 Campylobacter jejuni
2.4.99.18 PglB single-subunit membrane protein Campylobacter jejuni
2.4.99.19 oligosaccharyltransferase
-
 Campylobacter jejuni
2.4.99.19 OST
-
 Campylobacter jejuni
2.4.99.19 PglB
-
 Campylobacter jejuni

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.99.18 7.5
-
 assay at Campylobacter jejuni

General Information

EC Number General Information Comment Organism
2.4.99.19 evolution bacterial PglB and archaeal AglB constitute a protein family of the catalytic subunit of OST, along with STT3 from eukaryotes, that has three types of OST catalytic centers, structure analysis and comparison, overview Campylobacter jejuni
2.4.99.19 additional information the PglB structure reveals a distinct catalytic motif in the transmembrane region that contributes to the catalytic function, structure analysis, overview Campylobacter jejuni