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Literature summary for 3.2.1.97 extracted from
- Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum (2005), J. Biol. Chem., 280, 37415-37422.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expression in Escherichia coli BL21 | Bifidobacterium longum |
| expression in Escherichia coli, wild-type and mutants D601N, D605N, D612N, D655N, D665N, E679Q, D682N, D682A, D700N, E723Q, D762N, D762A, D789N, D789A, E822Q, E822A, E828Q, D839N, D864N, D898N, D1058N, D1106N, D1248N, E1276Q, E1302Q, E1350Q | Bifidobacterium longum |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D1058N | activity in cell-free extracts of Escherichia coli almost comparable to wild-type | Bifidobacterium longum |
| D1106N | 10-30% activity compared to wild-type | Bifidobacterium longum |
| D1106N | Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity | Bifidobacterium longum |
| D1248N | 10-30% activity compared to wild-type | Bifidobacterium longum |
| D601N | activity in cell-free extracts of Escherichia coli almost comparable to wild-type | Bifidobacterium longum |
| D601N | Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type | Bifidobacterium longum |
| D605N | 10-30% activity compared to wild-type | Bifidobacterium longum |
| D605N | Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity | Bifidobacterium longum |
| D612N | activity in cell-free extracts of Escherichia coli almost comparable to wild-type | Bifidobacterium longum |
| D612N | Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type | Bifidobacterium longum |
| D655N | 10-30% activity compared to wild-type | Bifidobacterium longum |
| D655N | Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity | Bifidobacterium longum |
| D665N | activity in cell-free extracts of Escherichia coli almost comparable to wild-type | Bifidobacterium longum |
| D665N | Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type | Bifidobacterium longum |
| D682A | no activity | Bifidobacterium longum |
| D682N | Escherichia coli cells expressing the mutant enzyme exhibit less than 2% of the wild-type endo-alpha-GalNAc activity | Bifidobacterium longum |
| D682N | significant decrease in activity (below 2%) | Bifidobacterium longum |
| D700N | activity in cell-free extracts of Escherichia coli almost comparable to wild-type | Bifidobacterium longum |
| D700N | Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type | Bifidobacterium longum |
| D762A | activity almost comparable to wild-type | Bifidobacterium longum |
| D762A | kcat/KM is 3.9fold lower than wild-type value | Bifidobacterium longum |
| D762N | Escherichia coli cells expressing the mutant enzyme exhibit less than 2% of the wild-type endo-alpha-GalNAc activity | Bifidobacterium longum |
| D762N | significant decrease in activity (below 2%) | Bifidobacterium longum |
| D789A | no activity | Bifidobacterium longum |
| D789N | Escherichia coli cells expressing the mutant enzyme exhibit less than 2% of the wild-type endo-alpha-GalNAc activity | Bifidobacterium longum |
| D789N | significant decrease in activity (below 2%) | Bifidobacterium longum |
| D839N | activity in cell-free extracts of Escherichia coli almost comparable to wild-type | Bifidobacterium longum |
| D839N | Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type | Bifidobacterium longum |
| D864N | activity in cell-free extracts of Escherichia coli almost comparable to wild-type | Bifidobacterium longum |
| D864N | Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type | Bifidobacterium longum |
| D898N | activity in cell-free extracts of Escherichia coli almost comparable to wild-type | Bifidobacterium longum |
| D898N | Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type | Bifidobacterium longum |
| E1276Q | 10-30% activity compared to wild-type | Bifidobacterium longum |
| E1276Q | Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity | Bifidobacterium longum |
| E1302Q | activity in cell-free extracts of Escherichia coli almost comparable to wild-type | Bifidobacterium longum |
| E1302Q | Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type | Bifidobacterium longum |
| E1350Q | activity in cell-free extracts of Escherichia coli almost comparable to wild-type | Bifidobacterium longum |
| E1350Q | Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type | Bifidobacterium longum |
| E679Q | activity in cell-free extracts of Escherichia coli almost comparable to wild-type | Bifidobacterium longum |
| E679Q | Escherichia coli cells expressing the mutant enzyme exhibit endo-alpha-GalNAc activity almost comparable with that of the cells carrying wild-type | Bifidobacterium longum |
| E723Q | 10-30% activity compared to wild-type | Bifidobacterium longum |
| E723Q | Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity | Bifidobacterium longum |
| E822A | kcat/KM is 380fold lower than wild-type value | Bifidobacterium longum |
| E822A | significant decrease in kcat and slight increase in Km | Bifidobacterium longum |
| E822Q | significant decrease in activity (below 2%) | Bifidobacterium longum |
| E828Q | 10-30% activity compared to wild-type | Bifidobacterium longum |
| E828Q | Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity | Bifidobacterium longum |
| E882Q | Escherichia coli cells expressing the mutant enzyme exhibit less than 2% of the wild-type endo-alpha-GalNAc activity | Bifidobacterium longum |
| S1248N | Escherichia coli cells expressing the mutant enzyme exhibit 10-30% of the wild-type endo-alpha-GalNAc activity | Bifidobacterium longum |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0218 | - |
Galbeta(1-3)GalNAcalpha1-p-nitrophenol | wild-type enzyme | Bifidobacterium longum |  |
| 0.0218 | - |
Galbeta(1,3)GalNAcalpha-1-p-nitrophenol | wild-type, pH 5.0, 37°C | Bifidobacterium longum | |
| 0.0384 | - |
Galbeta(1,3)GalNAcalpha-1-p-nitrophenol | mutant D762A, pH 5.0, 37°C | Bifidobacterium longum | |
| 0.0384 | - |
Galbeta(1-3)GalNAcalpha1-p-nitrophenol | mutant enzyme D762A | Bifidobacterium longum | |
| 0.107 | - |
Galbeta(1,3)GalNAcalpha-1-p-nitrophenol | mutant E822A, pH 5.0, 37°C | Bifidobacterium longum | |
| 0.107 | - |
Galbeta(1-3)GalNAcalpha1-p-nitrophenol | mutant enzyme E822A | Bifidobacterium longum | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 200000 | - |
gel filtration | Bifidobacterium longum |
| 210269 | - |
x * 210269, calculated from sequence | Bifidobacterium longum |
| 210300 | - |
calculated from amino acid sequence | Bifidobacterium longum |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-serine-[protein] + H2O | Bifidobacterium longum | - |
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-serine-[protein] | - |
? | |
| 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-serine-[protein] + H2O | Bifidobacterium longum JCM 1217 | - |
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-serine-[protein] | - |
? | |
| 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-threonine-[protein] + H2O | Bifidobacterium longum | - |
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-threonine-[protein] | - |
? | |
| 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-threonine-[protein] + H2O | Bifidobacterium longum JCM 1217 | - |
3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-threonine-[protein] | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Bifidobacterium longum | Q3T552 | - |
- |
| Bifidobacterium longum | Q3T552 | JCM 1217 | - |
| Bifidobacterium longum JCM 1217 | Q3T552 | - |
- |
| Bifidobacterium longum JCM 1217 | Q3T552 | JCM 1217 | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
- |
Bifidobacterium longum |
| recombinant | Bifidobacterium longum |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-serine-[protein] + H2O | - |
Bifidobacterium longum | 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-serine-[protein] | - |
? | |
| 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-serine-[protein] + H2O | - |
Bifidobacterium longum JCM 1217 | 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-serine-[protein] | - |
? | |
| 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-threonine-[protein] + H2O | - |
Bifidobacterium longum | 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-threonine-[protein] | - |
? | |
| 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosaminyl-L-threonine-[protein] + H2O | - |
Bifidobacterium longum JCM 1217 | 3-O-beta-D-galactosyl-N-acetyl-alpha-D-galactosamine + L-threonine-[protein] | - |
? | |
| asialofetuin + H2O | - |
Bifidobacterium longum | Galbeta(1-3)GalNAc + ? | - |
? | |
| asialofetuin + H2O | - |
Bifidobacterium longum JCM 1217 | Galbeta(1-3)GalNAc + ? | - |
? | |
| Galbeta(1,3)GalNAc-asialofetuin + H2O | pH 5.0, 37°C | Bifidobacterium longum | Galbeta(1,3)GalNAc + asialofetuin | - |
? | |
| Galbeta(1,3)GalNAcalpha-1-p-nitrophenol + H2O | pH 5.0, 37°C | Bifidobacterium longum | p-nitrophenol + Galbeta(1,3)GalNAc | - |
? | |
| Galbeta(1-3)GalNAcalpha1-p-nitrophenol + H2O | - |
Bifidobacterium longum | Galbeta(1-3)GalNAc + p-nitrophenol | - |
? | |
| additional information | no reaction with Galbeta(1,3)(GlcNAcbeta(1,6))GalNAcalpha-1-p-nitrophenol, GalNAcalpha-1-p-nitrophenol, GlcNAcbeta(1,3)GalNAcalpha-1-p-nitrophenol and Galbeta(1,3)GlcNAcalpha-1-p-nitrophenol | Bifidobacterium longum | ? | - |
? | |
| additional information | the enzyme also exhibits transglycosylation activity towards various mono- and disaccharides and 1-alkanols. No activity with GalNAc-p-nitrophenol, Galbeta(1-3)(GlcNAcbeta(1-6))GalNAc-p-nitrophenol, GlcNAcbeta(1-3)GalNAc-p-nitrophenol, Galbeta(1-3)GlcNAc-p-nitrophenol. Does not release sialo-oligosaccharides from fetuin | Bifidobacterium longum | ? | - |
? | |
| additional information | no reaction with Galbeta(1,3)(GlcNAcbeta(1,6))GalNAcalpha-1-p-nitrophenol, GalNAcalpha-1-p-nitrophenol, GlcNAcbeta(1,3)GalNAcalpha-1-p-nitrophenol and Galbeta(1,3)GlcNAcalpha-1-p-nitrophenol | Bifidobacterium longum JCM 1217 | ? | - |
? | |
| additional information | the enzyme also exhibits transglycosylation activity towards various mono- and disaccharides and 1-alkanols. No activity with GalNAc-p-nitrophenol, Galbeta(1-3)(GlcNAcbeta(1-6))GalNAc-p-nitrophenol, GlcNAcbeta(1-3)GalNAc-p-nitrophenol, Galbeta(1-3)GlcNAc-p-nitrophenol. Does not release sialo-oligosaccharides from fetuin | Bifidobacterium longum JCM 1217 | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| ? | x * 200000, SDS-PAGE | Bifidobacterium longum |
| ? | x * 210269, calculated from sequence | Bifidobacterium longum |
| monomer | 1 * 200000, SDS-PAGE, gel filtration | Bifidobacterium longum |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| endo-alpha-N-acetylgalactosaminidase | - |
Bifidobacterium longum |
| EngBF | - |
Bifidobacterium longum |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 60 | - |
- |
Bifidobacterium longum |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
stable up to | Bifidobacterium longum |
| 37 | - |
the enzyme is stable up to 37°C and retains 73% activity after incubation at 45°C for 30 min in 50 mM acetate buffer (pH 5.0) | Bifidobacterium longum |
| 45 | - |
pH 5.0, 30 min, enzyme retains 73% of initial activity | Bifidobacterium longum |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.23 | - |
Galbeta(1,3)GalNAcalpha-1-p-nitrophenol | mutant E822A, pH 5.0, 37°C | Bifidobacterium longum | |
| 0.23 | - |
Galbeta(1-3)GalNAcalpha1-p-nitrophenol | mutant enzyme E822A | Bifidobacterium longum | |
| 8.17 | - |
Galbeta(1,3)GalNAcalpha-1-p-nitrophenol | mutant D762A, pH 5.0, 37°C | Bifidobacterium longum | |
| 8.17 | - |
Galbeta(1-3)GalNAcalpha1-p-nitrophenol | mutant enzyme D762A | Bifidobacterium longum | |
| 17.8 | - |
Galbeta(1-3)GalNAcalpha1-p-nitrophenol | wild-type enzyme | Bifidobacterium longum | |
| 17.8 | - |
Galbeta(1,3)GalNAcalpha-1-p-nitrophenol | pH 5.0, 37°C | Bifidobacterium longum | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 5 | - |
- |
Bifidobacterium longum |
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