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Literature summary for 3.2.1.96 extracted from

  • Umekawa, M.; Li, C.; Higashiyama, T.; Huang, W.; Ashida, H.; Yamamoto, K.; Wang, L.X.
    Efficient glycosynthase mutant derived from Mucor hiemalis endo-beta-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan (2010), J. Biol. Chem., 285, 511-521.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type Endo-M and mutants in Escherichia coli strain BL21(DE3) Mucor hiemalis

Protein Variants

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Mucor hiemalis
-
-
-

Specific Activity [micromol/min/mg]

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 and Products (Substrate)

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

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 [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Mucor hiemalis

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.5
-
hydrolytic assay at Mucor hiemalis
7.2
-
transglycosylation assay at Mucor hiemalis

kcat/KM [mM/s]

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