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Literature summary for 3.2.1.123 extracted from

  • Han, Y.B.; Chen, L.Q.; Li, Z.; Tan, Y.M.; Feng, Y.; Yang, G.Y.
    Structural insights into the broad substrate specificity of a novel endoglycoceramidase I belonging to a new subfamily of GH5 glycosidases (2017), J. Biol. Chem., 292, 4789-4800 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.2.1.123

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli BL21 (DE3) Rhodococcus sp. M-777

Crystallization (Commentary)

Crystallization (Comment) Organism
hanging drop vapor diffusion method at 20°C, X-ray crystal structures of the enzyme alone and in complex with its substrates monosialodihexosylganglioside and monosialotetrahexosylganglioside determined at 2.11 A resolution. Compared with EGCase II from Rhodococcus sp. M-777, which possesses strict substrate specificity, EGCase I from Rhodococcus hoagii 103S possesses a longer alpha7-helix and a shorter loop 4, which forms a larger substrate-binding pocket that could accommodate more extended oligosaccharides. In addition, loop 2 and loop 8 of the enzyme adopt a more open conformation, which also enlarges the oligosaccharide-binding cavity Rhodococcus equi

Protein Variants

Protein Variants Comment Organism
D133A no hydrolysis of beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide and beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide Rhodococcus equi
D342A no hydrolysis of beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide and beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide Rhodococcus equi
D62A hydrolysis of beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide is 71.9% compared to wild-type enzyme. Hydrolysis of beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide is 83.9% compared to wild-type enzyme Rhodococcus equi
delN148-G154 loop-deleted mutant Asn148-Gly154, the truncation of loop 4 from Asn148 to Gly154 may increase the space of the crowded sugar-binding cavity, resulting in enhanced activity toward beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide (163%) and decreased activity (46.8%) toward beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide Rhodococcus sp. M-777
E339S no hydrolysis of beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide and beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide Rhodococcus equi
K61A no hydrolysis of beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide and beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide Rhodococcus equi
N213A no hydrolysis of beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide and beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide Rhodococcus equi
N265A no hydrolysis of beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide. Hydrolysis of beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide is 2.1% compared to wild-type enzyme Rhodococcus equi
Q298A no hydrolysis of beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide and beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide Rhodococcus equi
S63G the mutant enzyme shows a broader substrate spectrum and significantly increased activity toward bulky substrates Rhodococcus sp. M-777
W365A no hydrolysis of beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide and beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide Rhodococcus equi
Y302A no hydrolysis of beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide and beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide Rhodococcus equi

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.23
-
 beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide pH 6.0, 37°C Rhodococcus equi
0.25
-
 beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide pH 6.0, 37°C Rhodococcus sp. M-777

Organism

Organism UniProt Comment Textmining
Rhodococcus equi A0A3S5YBC7
-
-
Rhodococcus equi 103S A0A3S5YBC7
-
-
Rhodococcus sp. M-777 O33853
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.19
-
 substrate: beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide, pH 6.0, 37°C Rhodococcus sp. M-777
0.24
-
 substrate: beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide, pH 6.0, 37°C Rhodococcus sp. M-777
0.45
-
 substrate: beta-D-GalNAc-(1->3)-alpha-Gal-(1->4)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide, pH 6.0, 37°C Rhodococcus equi
0.61
-
 substrate: alpha-Neu5Ac-(2->3)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide, pH 6.0, 37°C Rhodococcus sp. M-777
1.8
-
 substrate: beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide, pH 6.0, 37°C Rhodococcus equi
3.21
-
 substrate: alpha-L-Fuc-(1->2)-beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide, pH 6.0, 37°C Rhodococcus equi
4.93
-
 substrate: alpha-Neu5Ac-(2->3)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide, pH 6.0, 37°C Rhodococcus equi
6.44
-
 substrate: beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide, pH 6.0, 37°C Rhodococcus equi

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
alpha-L-Fuc-(1->2)-beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O
-
 Rhodococcus equi ceramide + alpha-L-Fuc-(1->2)-beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc
-
 ?
alpha-L-Fuc-(1->2)-beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O
-
 Rhodococcus equi 103S ceramide + alpha-L-Fuc-(1->2)-beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc
-
 ?
alpha-Neu5Ac-(2->3)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O
-
 Rhodococcus equi ceramide + alpha-Neu5Ac-(2->3)-beta-D-Gal-(1->4)-Glc
-
 ?
alpha-Neu5Ac-(2->3)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O preferred substrate Rhodococcus sp. M-777 ceramide + alpha-Neu5Ac-(2->3)-beta-D-Gal-(1->4)-Glc
-
 ?
alpha-Neu5Ac-(2->3)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O
-
 Rhodococcus equi 103S ceramide + alpha-Neu5Ac-(2->3)-beta-D-Gal-(1->4)-Glc
-
 ?
beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O
-
 Rhodococcus equi ceramide + beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc
-
 ?
beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O
-
 Rhodococcus sp. M-777 ceramide + beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc
-
 ?
beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O
-
 Rhodococcus equi 103S ceramide + beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc
-
 ?
beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O
-
 Rhodococcus equi ceramide + beta-D-Gal-(1->4)-Glc
-
 ?
beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O
-
 Rhodococcus sp. M-777 ceramide + beta-D-Gal-(1->4)-Glc
-
 ?
beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O
-
 Rhodococcus equi 103S ceramide + beta-D-Gal-(1->4)-Glc
-
 ?
beta-D-GalNAc-(1->3)-alpha-Gal-(1->4)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide + H2O
-
 Rhodococcus equi ceramide + beta-D-GalNAc-(1->3)-alpha-Gal-(1->4)-beta-D-Gal-(1->4)-beta-D-Glc
-
 ?
additional information no activity with beta-D-Glc-(1<->1')-ceramide, beta-D-Gal-(1<->1')-ceramide, beta-D-GalNAc-(1->3)-alpha-Gal-(1->4)-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide and alpha-L-Fuc-(1->2)-beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide Rhodococcus sp. M-777 ?
-
 ?
additional information specifically hydrolyzes the glycosidic linkage between the oligosaccharide and the ceramide moieties of various glycosphingolipids. No activity with beta-D-Glc-(1<->1')-ceramide and beta-D-Gal-(1<->1')-ceramide Rhodococcus equi ?
-
 ?
additional information specifically hydrolyzes the glycosidic linkage between the oligosaccharide and the ceramide moieties of various glycosphingolipids. No activity with beta-D-Glc-(1<->1')-ceramide and beta-D-Gal-(1<->1')-ceramide Rhodococcus equi 103S ?
-
 ?

Synonyms

Synonyms Comment Organism
EGCase I
-
 Rhodococcus equi
EGCase II
-
 Rhodococcus sp. M-777
endoglycoceramidase I
-
 Rhodococcus equi
endoglycoceramidase II
-
 Rhodococcus sp. M-777

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.088
-
 beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide pH 6.0, 37°C Rhodococcus sp. M-777
10.5
-
 beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide pH 6.0, 37°C Rhodococcus equi

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.35
-
 beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide pH 6.0, 37°C Rhodococcus sp. M-777
45.7
-
 beta-D-Gal-(1->3)-beta-D-GalNAc-(1->4)-(alpha-Neu5Ac-(2->3))-beta-D-Gal-(1->4)-beta-D-Glc-(1<->1')-ceramide pH 6.0, 37°C Rhodococcus equi