Literature summary for 3.1.1.56 extracted from

Wu, D.; Li, Y.; Song, G.; Zhang, D.; Shaw, N.; Liu, Z.J.
Crystal structure of human esterase D: a potential genetic marker of retinoblastoma (2009), FASEB J., 23, 1441-1446.

Application

Application	Comment	Organism
diagnostics	assisting early retinoblastoma diagnosis	Homo sapiens
molecular biology	studying the physiological role	Homo sapiens

Cloned(Commentary)

Cloned (Comment)	Organism
from brain cDNA in pMCSG7 for inducible expression with N-terminal hexa-His-tag in Escherichia coli BL21(DE3)	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
hanging drop: 16°C, 3 days, reservoir solution: sodium citrate pH 5.6, 200 mM ammonium acetate, 30% (w/v) polyethylene glycol (PEG) 4000, crystal: space group P2(1), unit cell parameters: a: 51.54, b: 70.72, c: 65.01, alpha: 90°, beta: 108.84°, gamma: 90°, 2 molecules in the asymmetric unit, molecular replacement using PDB: 1PV1, co-crystallization and soaking with substrate failed, structure: several insertions in canonical alpha/beta-hydrolase fold, GxSxG motif, active site in positively charged cleft including residues S149, D226, and H260, and aromatic residues (H148, F172 W183, F228, H260, Y262) lined at the surface, catalysis mediated by residues S153, H264, and D230, oxyanion hole formed by e.g. L54 and M150	Homo sapiens

Crystallization (Comment)

Organism

hanging drop: 16°C, 3 days, reservoir solution: sodium citrate pH 5.6, 200 mM ammonium acetate, 30% (w/v) polyethylene glycol (PEG) 4000, crystal: space group P2(1), unit cell parameters: a: 51.54, b: 70.72, c: 65.01, alpha: 90°, beta: 108.84°, gamma: 90°, 2 molecules in the asymmetric unit, molecular replacement using PDB: 1PV1, co-crystallization and soaking with substrate failed, structure: several insertions in canonical alpha/beta-hydrolase fold, GxSxG motif, active site in positively charged cleft including residues S149, D226, and H260, and aromatic residues (H148, F172 W183, F228, H260, Y262) lined at the surface, catalysis mediated by residues S153, H264, and D230, oxyanion hole formed by e.g. L54 and M150

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
D226A	4.3% of wild-type activity	Homo sapiens
D226N	9% of wild-type activity	Homo sapiens
H260A	3% of wild-type activity	Homo sapiens
H260Q	1.3% of wild-type activity	Homo sapiens
L54A	83% of wild-type activity	Homo sapiens
M150	162% of wild-type activity, possibly due to reduced steric hindrance	Homo sapiens
S149A	catalytically inactive	Homo sapiens
S149T	1.3% of wild-type activity	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
4-methylumbelliferyl acetate + H2O	Homo sapiens	hydrolysis via formation of covalent, negatively charged enzyme-substrate tetrahydral intermediate: nucleophilic attack of S149 on carbonyl moiety after activation by H260, intermediate stabilized in oxyanion hole by M150 and L54, H260 stabilized by D226, release of alcohol followed by nucleophilic attack of water molecule at the acidic moiety and release of acetate	4-methylumbelliferone + acetate	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
by nickel-affinity chromatography (elution: 300 mM imidazole) followed by cleavage of His-tag by tobacco etch virus (TEV) protease, a second nickel-affinity chromatography, and size-exclusion chromatography (Superdex G75) in presence of 1 mM dithiothreitol	Homo sapiens

Source Tissue

Source Tissue	Comment	Organism	Textmining
brain	-	Homo sapiens	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
4-methylumbelliferyl acetate + H2O	hydrolysis via formation of covalent, negatively charged enzyme-substrate tetrahydral intermediate: nucleophilic attack of S149 on carbonyl moiety after activation by H260, intermediate stabilized in oxyanion hole by M150 and L54, H260 stabilized by D226, release of alcohol followed by nucleophilic attack of water molecule at the acidic moiety and release of acetate	Homo sapiens	4-methylumbelliferone + acetate	-	?
4-methylumbelliferyl acetate + H2O	2 min, 25°C, pH 7.4	Homo sapiens	4-methylumbelliferone + acetate	reaction stop by citric acid, quantification at 460 nm	?

Synonyms

Synonyms	Comment	Organism
ESD	-	Homo sapiens
esterase D	-	Homo sapiens
S-formylglutathione hydrolase	-	Homo sapiens