Literature summary for 3.2.1.123 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.

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Application

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

Cloned(Commentary)

Cloned (Comment)	Organism
subcloned into pET28a using the NdeI/XhoI restriction sites and expressed in Escherichia coli BL21	Rhodococcus sp.

Crystallization (Commentary)

Crystallization (Comment)	Organism
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.

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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.
E351S	prevents substrate hydrolysis during crystallization	Rhodococcus sp.

KM Value [mM]

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

Localization

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

Organism

Organism	UniProt	Comment	Textmining
Rhodococcus sp.	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
by Ni(II) affinity chromatography to more than 95% purity	Rhodococcus sp.

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
D-erythro-dihydrosphingosine + H2O	-	Rhodococcus sp.	?	-	?
D-erythro-sphingosine + H2O	-	Rhodococcus sp.	?	-	?
ganglioside + H2O	-	Rhodococcus sp.	?	-	?

Synonyms

Synonyms	Comment	Organism
EGC	-	Rhodococcus sp.
endo-glycoceramidase II	-	Rhodococcus sp.

Turnover Number [1/s]

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