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Literature summary extracted from

  • Caines, M.E.; Vaughan, M.D.; Tarling, C.A.; Hancock, S.M.; Warren, R.A.; Withers, S.G.; Strynadka, N.C.
    Structural and mechanistic analyses of endo-glycoceramidase II, a membrane-associated family 5 glycosidase in the Apo and GM3 ganglioside-bound forms (2007), J. Biol. Chem., 282, 14300-14308.
    View publication on PubMed

Application

EC Number Application Comment Organism
3.2.1.123 drug development rational redesign of EGC toward the synthesis of novel ganglioside-derived therapeutics Rhodococcus sp.

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.62 expression of the gene lacking the 30-residue N-terminal secretion signal sequence in Escherichia coli, selenomethionine-derivatized enzyme Rhodococcus sp.
3.2.1.123 subcloned into pET28a using the NdeI/XhoI restriction sites and expressed in Escherichia coli BL21 Rhodococcus sp.

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.2.1.62 by vapor diffusion in the presence of detergents like Triton X-100, octyl beta-D-glucopyranoside, decyl beta-D-maltopyranoside, and dodecyl beta-D-maltopyranoside, form, in complex with the ganglioside GM3 Rhodococcus sp.
3.2.1.123 by vapor diffusion method, crystals of the native enzyme belong to space group P21 with unit cell dimensions a=53.8 A, b=92.9 Å, c=94.5 A,beta=98.6°, to 1.6 A resolution. Crystal structure of mutant E351S in complex with the ganglioside GM3, crystals belong to space group C2, with unit cell dimensions a = 77.8 A, b = 62.0 A, c = 102.8 A, beta = 112.3°, to 1.1 A resolution. Active site of endo-glycoceramidase is split into a wide, polar cavity to bind the polyhydroxylated oligosaccharide moiety and a narrow, hydrophobic tunnel to bind the ceramide lipid chains Rhodococcus sp.

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.62 E233A site directed mutagenesis Rhodococcus sp.
3.2.1.62 E351S site directed mutagenesis Rhodococcus sp.
3.2.1.123 E233A glycosyl-enzyme intermediate trapped by soaking crystals of the general acid/base knock-out mutant E233A, with an activated sugar donor, 2,4-dinitrophenyl beta-D-lactoside Rhodococcus sp.
3.2.1.123 E351S prevents substrate hydrolysis during crystallization Rhodococcus sp.

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.62 0.04
-
 D-erythro-sphingosine
-
 Rhodococcus sp.
3.2.1.62 0.3
-
 D-erythro-dihydrosphingosine
-
 Rhodococcus sp.
3.2.1.123 0.04
-
 D-erythro-sphingosine
-
 Rhodococcus sp.
3.2.1.123 0.3
-
 D-erythro-dihydrosphingosine
-
 Rhodococcus sp.

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
3.2.1.123 membrane
-
 Rhodococcus sp. 16020
-

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.62 Rhodococcus sp.
-
-
-
3.2.1.123 Rhodococcus sp.
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.62 of the recombinant protein by by Ni(II) affinity chromatography Rhodococcus sp.
3.2.1.123 by Ni(II) affinity chromatography to more than 95% purity Rhodococcus sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.62 D-erythro-dihydrosphingosine + a sugar
-
 Rhodococcus sp. ?
-
 ?
3.2.1.62 D-erythro-sphingosine + a sugar
-
 Rhodococcus sp. ?
-
 ?
3.2.1.123 D-erythro-dihydrosphingosine + H2O
-
 Rhodococcus sp. ?
-
 ?
3.2.1.123 D-erythro-sphingosine + H2O
-
 Rhodococcus sp. ?
-
 ?
3.2.1.123 ganglioside + H2O
-
 Rhodococcus sp. ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.2.1.62 EGC
-
 Rhodococcus sp.
3.2.1.62 endo-glycoceramidase II membrane-associated family 5 glycosidase Rhodococcus sp.
3.2.1.62 glycosylceramidase
-
 Rhodococcus sp.
3.2.1.123 EGC
-
 Rhodococcus sp.
3.2.1.123 endo-glycoceramidase II
-
 Rhodococcus sp.

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.123 0.07
-
 D-erythro-sphingosine
-
 Rhodococcus sp.
3.2.1.123 0.12
-
 D-erythro-dihydrosphingosine
-
 Rhodococcus sp.