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 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 | 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 |
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 |
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 |
EC Number | Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|---|
2.4.99.18 | membrane | - |
Campylobacter jejuni | 16020 | - |
2.4.99.19 | membrane | - |
Campylobacter jejuni | 16020 | - |
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 | - |
? |
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 | - |
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 |
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 | - |
? |
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 |
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 |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
2.4.99.18 | 7.5 | - |
assay at | Campylobacter jejuni |
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 |