Literature summary for 3.2.1.96 extracted from

Yin, J.; Li, L.; Shaw, N.; Li, Y.; Song, J.K.; Zhang, W.; Xia, C.; Zhang, R.; Joachimiak, A.; Zhang, H.C.; Wang, L.X.; Liu, Z.J.; Wang, P.
Structural basis and catalytic mechanism for the dual functional endo-beta-N-acetylglucosaminidase A (2009), PLoS ONE, 4, e4658.

Cloned(Commentary)

Cloned (Comment)	Organism
into vector pET15b and transformed into Escherichia coli BL21(DE3) cells	Glutamicibacter protophormiae

Crystallization (Commentary)

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

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

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

Inhibitors	Comment	Organism	Structure
Manalpha(1-6)(Manalpha(1-3))Manbeta(1-4)GlcNAc-thiazoline	-	Glutamicibacter protophormiae

Organism

Organism	UniProt	Comment	Textmining
Glutamicibacter protophormiae	Q9ZB22	-	-

Purification (Commentary)

Purification (Comment)	Organism
on Ni-NTA resin and on ion-exchange chromatography column and by gel filtration	Glutamicibacter protophormiae

Specific Activity [micromol/min/mg]

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 and Products (Substrate)

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

Synonyms	Comment	Organism
Endo-A	-	Glutamicibacter protophormiae
endo-beta-N-acetylglucosaminidase A	-	Glutamicibacter protophormiae
ENGase	-	Glutamicibacter protophormiae