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Literature summary for 2.7.7.83 extracted from
- Improvement of ST0452 N-acetylglucosamine-1-phosphate uridyltransferase activity by the cooperative effect of two single mutations identified through structure-based protein engineering (2018), Appl. Environ. Microbiol., 84, e02213-18 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant expression of His-tagged wild-type enzyme and mutant Y97N in Escherichia coli strain BL21-CodonPlus(DE3)-RIPL | Sulfurisphaera tokodaii |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| purified recombinant enzyme mutant Y97N in complex with UTP or N-acetyl-alpha-D-glucosamine 1-phosphate, sitting drop vapor diffusion method, mixing of 11 mg/ml protein with ligands 5 mM UDP-GlcNAc and 10 mM (NH4)2SO4 (to form the complex with UDP-GlcNAc) or 5 mM UTP and 10 mM (NH4)2SO4 (to form the complex with UTP), and with cyrstallization solution containing 20% w/v PEG 3350 and 0.2 M potassium citrate tribasic monohydrate, X-ray diffraction structure determination and analysis at 2.91 and 2.09 A resolution, respectively | Sulfurisphaera tokodaii |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| additional information | all proteins substituted at position 146 have drastically decreased activities, whereas several proteins substituted at position 80 show higher GlcNAc-1-P UTase activity, compared to that of the wild-type protein | Sulfurisphaera tokodaii |
| T80A | site-directed mutagenesis, the mutant shows increased GlcNAc-1-P and Glc-1-P UTase activity compared to wild-type | Sulfurisphaera tokodaii |
| T80C | site-directed mutagenesis, the mutant shows slightly reduced GlcNAc-1-P UTase activity compared to wild-type | Sulfurisphaera tokodaii |
| T80D | site-directed mutagenesis, the mutant shows increased Glc-1-P UTase activity and reduced GlcNAc-1-P UTase activity compared to wild-type | Sulfurisphaera tokodaii |
| T80E | site-directed mutagenesis, the mutant shows no Glc-1-P UTase activity and highly reduced GlcNAc-1-P UTase activity | Sulfurisphaera tokodaii |
| T80F | site-directed mutagenesis, the mutant shows no and Glc-1-P UTase and GlcNAc-1-P UTase activity | Sulfurisphaera tokodaii |
| T80G | site-directed mutagenesis, the mutant shows increased GlcNAc-1-P and Glc-1-P UTase activity compared to wild-type | Sulfurisphaera tokodaii |
| T80H | site-directed mutagenesis, the mutant shows increased Glc-1-P UTase activity and reduced GlcNAc-1-P UTase activity compared to wild-type | Sulfurisphaera tokodaii |
| T80I | site-directed mutagenesis, the mutant shows no Glc-1-P UTase and GlcNAc-1-P UTase activity | Sulfurisphaera tokodaii |
| T80K | site-directed mutagenesis, the mutant shows no Glc-1-P UTase activity and highly reduced GlcNAc-1-P UTase activity | Sulfurisphaera tokodaii |
| T80L | site-directed mutagenesis, the mutant shows reduced GlcNAc-1-P UTase activity and no Glc-1-P UTase activity | Sulfurisphaera tokodaii |
| T80M | site-directed mutagenesis, the mutant shows reduced GlcNAc-1-P UTase activity and no Glc-1-P UTase activity | Sulfurisphaera tokodaii |
| T80N | site-directed mutagenesis, the mutant shows increased GlcNAc-1-P UTase activity compared to wild-type | Sulfurisphaera tokodaii |
| T80P | site-directed mutagenesis, the mutant shows slightly reduced GlcNAc-1-P UTase activity compared to wild-type | Sulfurisphaera tokodaii |
| T80Q | site-directed mutagenesis, the mutant shows increased GlcNAc-1-P and Glc-1-P UTase activity compared to wild-type | Sulfurisphaera tokodaii |
| T80R | site-directed mutagenesis, the mutant shows no Glc-1-P UTase activity and highly reduced GlcNAc-1-P UTase activity | Sulfurisphaera tokodaii |
| T80S | site-directed mutagenesis, the mutant shows the mutant shows increased GlcNAc-1-P UTase activity compared to wild-type | Sulfurisphaera tokodaii |
| T80S/Y97N | site-directed mutagenesis, the mutant shows 6.5times higher activity with N-acetylglucosamine-1-phosphate compared to that of the wild-type ST0452 protein | Sulfurisphaera tokodaii |
| T80V | site-directed mutagenesis, the mutant shows slightly reduced GlcNAc-1-P UTase activity compared to wild-type | Sulfurisphaera tokodaii |
| T80W | site-directed mutagenesis, the mutant shows no Glc-1-P UTase activity and highly reduced GlcNAc-1-P UTase activity | Sulfurisphaera tokodaii |
| T80Y | site-directed mutagenesis, the mutant shows no Glc-1-P UTase activity and highly reduced GlcNAc-1-P UTase activity | Sulfurisphaera tokodaii |
| Y97N | naturally occuring mutation, the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii, exhibits over 4times higher N-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase, EC 2.7.7.23) activity, compared with that of the wild-type ST0452 protein, three-dimensional structure analysis of the Y97N protein. The overall structure is almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions | Sulfurisphaera tokodaii |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Sulfurisphaera tokodaii |  |
| additional information | the three-dimensional structure of the ST0452 mutant Y97N is not changed by due to lack of metals but the interactions with the substrate is slightly modified, which might cause the activity to increase | Sulfurisphaera tokodaii |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| UTP + N-acetyl-alpha-D-galactosamine 1-phosphate | Sulfurisphaera tokodaii | - |
diphosphate + UDP-N-acetyl-alpha-D-galactosamine | - |
? | |
| UTP + N-acetyl-alpha-D-galactosamine 1-phosphate | Sulfurisphaera tokodaii 7 | - |
diphosphate + UDP-N-acetyl-alpha-D-galactosamine | - |
? | |
| UTP + N-acetyl-alpha-D-galactosamine 1-phosphate | Sulfurisphaera tokodaii DSM 16993 | - |
diphosphate + UDP-N-acetyl-alpha-D-galactosamine | - |
? | |
| UTP + N-acetyl-alpha-D-galactosamine 1-phosphate | Sulfurisphaera tokodaii JCM 10545 | - |
diphosphate + UDP-N-acetyl-alpha-D-galactosamine | - |
? | |
| UTP + N-acetyl-alpha-D-galactosamine 1-phosphate | Sulfurisphaera tokodaii NBRC 100140 | - |
diphosphate + UDP-N-acetyl-alpha-D-galactosamine | - |
? | |
| UTP + N-acetyl-alpha-D-glucosamine 1-phosphate | Sulfurisphaera tokodaii | reaction of EC 2.7.7.23 | diphosphate + UDP-N-acetyl-alpha-D-glucosamine | - |
? | |
| UTP + N-acetyl-alpha-D-glucosamine 1-phosphate | Sulfurisphaera tokodaii 7 | reaction of EC 2.7.7.23 | diphosphate + UDP-N-acetyl-alpha-D-glucosamine | - |
? | |
| UTP + N-acetyl-alpha-D-glucosamine 1-phosphate | Sulfurisphaera tokodaii DSM 16993 | reaction of EC 2.7.7.23 | diphosphate + UDP-N-acetyl-alpha-D-glucosamine | - |
? | |
| UTP + N-acetyl-alpha-D-glucosamine 1-phosphate | Sulfurisphaera tokodaii JCM 10545 | reaction of EC 2.7.7.23 | diphosphate + UDP-N-acetyl-alpha-D-glucosamine | - |
? | |
| UTP + N-acetyl-alpha-D-glucosamine 1-phosphate | Sulfurisphaera tokodaii NBRC 100140 | reaction of EC 2.7.7.23 | diphosphate + UDP-N-acetyl-alpha-D-glucosamine | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Sulfurisphaera tokodaii | Q975F9 | Sulfolobus tokodaii | - |
| Sulfurisphaera tokodaii 7 | Q975F9 | Sulfolobus tokodaii | - |
| Sulfurisphaera tokodaii DSM 16993 | Q975F9 | Sulfolobus tokodaii | - |
| Sulfurisphaera tokodaii JCM 10545 | Q975F9 | Sulfolobus tokodaii | - |
| Sulfurisphaera tokodaii NBRC 100140 | Q975F9 | Sulfolobus tokodaii | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type enzyme and mutant Y97N from Escherichia coli strain BL21-CodonPlus(DE3)-RIPL by nickel affinity chromatography and ultrafiltration | Sulfurisphaera tokodaii |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| UTP + alpha-D-glucose 1-phosphate | - |
Sulfurisphaera tokodaii | diphosphate + UDP-alpha-D-glucose | - |
? | |
| UTP + N-acetyl-alpha-D-galactosamine 1-phosphate | - |
Sulfurisphaera tokodaii | diphosphate + UDP-N-acetyl-alpha-D-galactosamine | - |
? | |
| UTP + N-acetyl-alpha-D-galactosamine 1-phosphate | - |
Sulfurisphaera tokodaii 7 | diphosphate + UDP-N-acetyl-alpha-D-galactosamine | - |
? | |
| UTP + N-acetyl-alpha-D-galactosamine 1-phosphate | - |
Sulfurisphaera tokodaii DSM 16993 | diphosphate + UDP-N-acetyl-alpha-D-galactosamine | - |
? | |
| UTP + N-acetyl-alpha-D-galactosamine 1-phosphate | - |
Sulfurisphaera tokodaii JCM 10545 | diphosphate + UDP-N-acetyl-alpha-D-galactosamine | - |
? | |
| UTP + N-acetyl-alpha-D-galactosamine 1-phosphate | - |
Sulfurisphaera tokodaii NBRC 100140 | diphosphate + UDP-N-acetyl-alpha-D-galactosamine | - |
? | |
| UTP + N-acetyl-alpha-D-glucosamine 1-phosphate | reaction of EC 2.7.7.23 | Sulfurisphaera tokodaii | diphosphate + UDP-N-acetyl-alpha-D-glucosamine | - |
? | |
| UTP + N-acetyl-alpha-D-glucosamine 1-phosphate | reaction of EC 2.7.7.23 | Sulfurisphaera tokodaii 7 | diphosphate + UDP-N-acetyl-alpha-D-glucosamine | - |
? | |
| UTP + N-acetyl-alpha-D-glucosamine 1-phosphate | reaction of EC 2.7.7.23 | Sulfurisphaera tokodaii DSM 16993 | diphosphate + UDP-N-acetyl-alpha-D-glucosamine | - |
? | |
| UTP + N-acetyl-alpha-D-glucosamine 1-phosphate | reaction of EC 2.7.7.23 | Sulfurisphaera tokodaii JCM 10545 | diphosphate + UDP-N-acetyl-alpha-D-glucosamine | - |
? | |
| UTP + N-acetyl-alpha-D-glucosamine 1-phosphate | reaction of EC 2.7.7.23 | Sulfurisphaera tokodaii NBRC 100140 | diphosphate + UDP-N-acetyl-alpha-D-glucosamine | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| GalNAc-1-P UTase | - |
Sulfurisphaera tokodaii |
| More | see also EC 2.7.7.23 | Sulfurisphaera tokodaii |
| ST0452 | - |
Sulfurisphaera tokodaii |
| STK_04520 | - |
Sulfurisphaera tokodaii |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 80 | - |
assay at | Sulfurisphaera tokodaii |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
assay at | Sulfurisphaera tokodaii |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | analysis of the overall structure of wild-type ST0452 protein (PDB ID 2GGO), residue 97 (Asn) interacts with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. UTP forms hydrogen bond interactions with seven residues, i.e. the main chain atoms of the position 8 Ala, position 9 Gly, position 12 Glu, position 79 Gly, and position 98 Gly residues and the side chain atoms of the position 13 Arg and position 73 Gln residues. The position 13 Arg and position 73 Gln residues appear to form more stable interactions than the other residues, with the position 13 Arg residue forming two hydrogen bonds with the phosphoryl group at the gamma-site and the amide group of the position 73 Gln residue forming a salt bridge with the uracil nucleobase in UTP | Sulfurisphaera tokodaii |
| physiological function | enzyme ST0452 is multifunctional | Sulfurisphaera tokodaii |
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