Cloned (Comment) | Organism |
---|---|
into vector pET15b and transformed into Escherichia coli BL21(DE3) cells | Glutamicibacter protophormiae |
Crystallization (Comment) | Organism |
---|---|
Endo-A in the native form or in complex with Man3GlcNAc-thiazoline or GlcNAc-Asn, between 2-3.5 A resolution. Crystals of Endo-A belong to P1 space group with four molecules of the protein in the asymmetric unit. The carbohydrate moiety sits above the TIM-barrel in a cleft region surrounded by aromatic residues. N171 is hydrogen bonded to the thiazoline nitrogen, mimicking the ability of the asparagine to orient the acetamido group for a nucleophilic attack on the anomeric carbon | Glutamicibacter protophormiae |
Protein Variants | Comment | Organism |
---|---|---|
N171A | completely abolishes enzymatic activity | Glutamicibacter protophormiae |
Y205F | exhibits reduced hydrolysis activity with an increase in transglycosylation yields | Glutamicibacter protophormiae |
Y299F | 3fold increase in the transglycosylation activity, while the hydrolysis activity remains unchanged | Glutamicibacter protophormiae |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
Manalpha(1-6)(Manalpha(1-3))Manbeta(1-4)GlcNAc-thiazoline | - |
Glutamicibacter protophormiae |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Glutamicibacter protophormiae | Q9ZB22 | - |
- |
Purification (Comment) | Organism |
---|---|
on Ni-NTA resin and on ion-exchange chromatography column and by gel filtration | Glutamicibacter protophormiae |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.01 | - |
specific transglycosylation activity of mutant N171A | Glutamicibacter protophormiae |
1.61 | - |
specific transglycosylation activity of mutant Y205F | Glutamicibacter protophormiae |
1.72 | - |
specific transglycosylation activity of the wild-type | Glutamicibacter protophormiae |
5.11 | - |
specific transglycosylation activity of mutant Y299F | Glutamicibacter protophormiae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
GlcNAc-Asn + Manalpha(1-6)(Manalpha(1-3))Manbeta(1-4)GlcNAc | Manalpha(1-6)(Manalpha(1-3))Manbeta(1-4)GlcNAc-thiazoline is a transition state mimic and is resistant to Endo-A hydrolysis | Glutamicibacter protophormiae | Manalpha(1-6)(Manalpha(1-3))Manbeta(1-4)GlcNAcbeta(1-4)GlcNAc-Asn | - |
? | |
additional information | conserved essential catalytic residues E173, N171 and Y205 are within hydrogen bonding distance of the substrate. W216 and W244 regulate access to the active site during transglycosylation by serving as gate-keepers | Glutamicibacter protophormiae | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
Endo-A | - |
Glutamicibacter protophormiae |
endo-beta-N-acetylglucosaminidase A | - |
Glutamicibacter protophormiae |
ENGase | - |
Glutamicibacter protophormiae |