Cloned (Comment) | Organism |
---|---|
expression of wild-type Endo-M and mutants in Escherichia coli strain BL21(DE3) | Mucor hiemalis |
Protein Variants | Comment | Organism |
---|---|---|
D147A | site-directed mutagenesis, the mutant show 19% reduced activity compared to the wild-type enzyme | Mucor hiemalis |
D158A | site-directed mutagenesis, the mutant show 88% reduced activity compared to the wild-type enzyme | Mucor hiemalis |
D170A | site-directed mutagenesis, the mutant show 98.8% reduced activity compared to the wild-type enzyme | Mucor hiemalis |
D218A | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
D279A | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
E177A | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
E177H | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
E177Q | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
additional information | as to the specific hydrolysis activity for N-glycopeptide SGP, i.e. biantennary complex-type sialylglycopeptide, all the Asn175 mutants show none or only marginal activity, but most of the Asn175 mutants show transglycosylation activity with Man9GlcNAc-oxazoline | Mucor hiemalis |
N175A | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
N175D | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
N175H | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
N175M | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
N175Q | site-directed mutagenesis, the mutant possesses dramatically enhanced glycosynthase-like activity with sugar oxazoline in comparison with N175A and a transglycosidase-like activity with natural N-glycan as well | Mucor hiemalis |
N175Q | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
N175Q/Y217F | site-directed mutagenesis, the double mutant is a glycosynthase, but the additional mutation at Tyr217 does not further enhance the activity | Mucor hiemalis |
N175S | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
N175T | site-directed mutagenesis, the mutant shows only residual hydrolytic activity | Mucor hiemalis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetic analysis, kinetics for the transglycosylation using Man9GlcNAc-oxazoline as a donor substrate, overview | Mucor hiemalis | |
3.5 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant N175Q | Mucor hiemalis | |
4.2 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant N175G | Mucor hiemalis | |
9.1 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant Y217F/N175Q | Mucor hiemalis | |
52 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant D279A | Mucor hiemalis | |
77.4 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant N175A | Mucor hiemalis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | Mucor hiemalis | Endo-M is unique in that it can transfer en bloc the oligosaccharide of various types of N-glycans onto different acceptors, and thereby it enzymatically generates diverse glycoconjugates | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Mucor hiemalis | - |
- |
- |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
N175 mutant hydrolytic activities, and that of mutants E177A, E177Q, E177H, D218A, and D279A lay below 1% of wild-type activity, overview | Mucor hiemalis |
9.5 | - |
purified recombinant wild-type enzyme, hydrolysis of natural N-glycan substrate | Mucor hiemalis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-nitrophenyl beta-N-acetyl-D-glucosamine + H2O | - |
Mucor hiemalis | 4-nitrophenol + N-acetyl-D-glucosamine | - |
? | |
additional information | Endo-M is unique in that it can transfer en bloc the oligosaccharide of various types of N-glycans onto different acceptors, and thereby it enzymatically generates diverse glycoconjugates | Mucor hiemalis | ? | - |
? | |
additional information | Endo-M can transfer en bloc the oligosaccharide of various types of N-glycans onto different acceptors, using synthetic sugar oxazoline or biantennary complex-type sialylglycopeptide as donor substrates, and thereby it enzymatically generates diverse glycoconjugates, key residue is the catalytic Asn175. Most of the Asn175 mutants have significantly diminished hydrolysis activity but act as glycosynthases capable of using synthetic sugar oxazoline for transglycosylation | Mucor hiemalis | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
endo-beta-N-acetylglucosaminidase | - |
Mucor hiemalis |
endo-M | - |
Mucor hiemalis |
ENGase | - |
Mucor hiemalis |
More | the enzyme belongs to the glycoside hydrolase family 85, GH85 | Mucor hiemalis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Mucor hiemalis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
11 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant N175A | Mucor hiemalis | |
64 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant N175G | Mucor hiemalis | |
67 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant Y217F/N175Q | Mucor hiemalis | |
150 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant D279A | Mucor hiemalis | |
250 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant N175Q | Mucor hiemalis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6.5 | - |
hydrolytic assay at | Mucor hiemalis |
7.2 | - |
transglycosylation assay at | Mucor hiemalis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.023 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant N175A | Mucor hiemalis | |
0.126 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant Y217F/N175Q | Mucor hiemalis | |
0.27 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant N175G | Mucor hiemalis | |
1.2 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant N175Q | Mucor hiemalis | |
1.28 | - |
4-nitrophenyl-beta-D-N-acetylglucosamine | pH 6.5, 30°C, recombinant mutant D279A | Mucor hiemalis |