Literature summary for 3.1.1.41 extracted from

Levisson, M.; Han, G.W.; Deller, M.C.; Xu, Q.; Biely, P.; Hendriks, S.; Ten Eyck, L.F.; Flensburg, C.; Roversi, P.; Miller, M.D.; McMullan, D.; von Delft, F.; Kreusch, A.; Deacon, A.M.; van der Oost, J.; Lesley, S.A.; Elsliger, M.A.; Kengen, S.W.; Wilson, I.A.
Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima (2012), Proteins, 80, 1545-1559.

Cloned(Commentary)

Cloned (Comment)	Organism
gene TM0077, expression of N-terminally His-tagged wild-type enzyme in Escherichia coli and selenomethionine-labeled enzyme in Escherichia coli methionine auxotrophic strain DL41	Thermotoga maritima

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant selenomethionine-substituted and wild-type enzyme in complex with covalently bound phenylmethylsulfonyl fluoride and paraoxon, hanging drop vapour diffusion method, for selenomethionine-substituted enzyme: mixing of 500 nl of 15 mg/ml protein in 20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP, in presence or absence of inhibitors, with 500 nl of reservoir solution containing 20% w/v PEG-3000, 0.1 M HEPES pH 7.5, 0.2 M NaCl, and equilibration against 0.25 ml of reservoir solution, for wild-type enzyme: mixing of 100 nl of 22.8 mg/ml protein in 20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP, in presence or absence of inhibitors, with 100 nl of reservoir solution containing 0.2 M calcium acetate hydrate, 20% w/v PEG 3350, pH 7.3, and equilibration against 0.06 ml of reservoir solution, 20°C, X-ray diffraction structure determination and analysis at 2.4 A and 2.1 A resolution, respectively, molecular replacement, structure modeling	Thermotoga maritima

Crystallization (Comment)

Organism

purified recombinant selenomethionine-substituted and wild-type enzyme in complex with covalently bound phenylmethylsulfonyl fluoride and paraoxon, hanging drop vapour diffusion method, for selenomethionine-substituted enzyme: mixing of 500 nl of 15 mg/ml protein in 20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP, in presence or absence of inhibitors, with 500 nl of reservoir solution containing 20% w/v PEG-3000, 0.1 M HEPES pH 7.5, 0.2 M NaCl, and equilibration against 0.25 ml of reservoir solution, for wild-type enzyme: mixing of 100 nl of 22.8 mg/ml protein in 20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP, in presence or absence of inhibitors, with 100 nl of reservoir solution containing 0.2 M calcium acetate hydrate, 20% w/v PEG 3350, pH 7.3, and equilibration against 0.06 ml of reservoir solution, 20°C, X-ray diffraction structure determination and analysis at 2.4 A and 2.1 A resolution, respectively, molecular replacement, structure modeling

Thermotoga maritima

Inhibitors

Inhibitors	Comment	Organism	Structure
paraoxon	competitive irreversible inhibitors of esterases. Inhibition proceeds by the formation of a reversible Michaelis complex, followed by an irreversible step, the inhibitor binds covalently to the catalytic serine (Ser188), upon binding of inhibitor, the catalytic serine adopts an altered conformation	Thermotoga maritima
phenylmethylsulfonyl fluoride	competitive irreversible inhibitors of esterases. Inhibition proceeds by the formation of a reversible Michaelis complex, followed by an irreversible step, the inhibitor binds covalently to the catalytic serine (Ser188), upon binding of inhibitor, the catalytic serine adopts an altered conformation. In the PMSF-bound structure, the phenyl ring of the inhibitor is located in the small active site groove surrounded by hydrophobic residues Tyr92, Trp124, Pro228, Ile276, and His303. The sulfonyl group of PMSF makes hydrogen bonds with the backbone amides of Tyr92 and Gln189	Thermotoga maritima

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
cytosol	the enzyme has no predicted signal sequence	Thermotoga maritima	5829	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	not required for activity, one calcium ion is located at the N-terminal region of helix alphaA-1, and is coordinated by the backbone carbonyl of Lys22 and the Glu26 carboxylate, Asp25 carboxylate contributes to the calcium binding via one of the coordinating water molecules. Another calcium ion is bound in a crystal packing interface between chain A and chain C' of a crystallographic symmetry-related hexamer and is coordinated by the carboxylates of GluA45 and AspA58 from one chain and the carboxylate from Glu C'45 (bidentate coordination) of the symmetry-related chain with three water molecules completing a capped-octahedral coordination sphere	Thermotoga maritima
additional information	no significant stimulation or reduction of enzyme activity in the presence of divalent metal ions or EDTA. In the paraoxon-bound structure, the diethylphosphate moiety is stabilized by hydrogen-bonding interactions with the oxyanion hole. One of the two ethyl arms of bound paraoxon points toward the larger pocket in the protein, while the other follows the groove of the small pocket. The two ethyl arms are stabilized by packing against Tyr92, Trp124, Pro228, Ile276, and His303	Thermotoga maritima

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
cephalosporin C + H2O	Thermotoga maritima	-	deacetylcephalosporin C + acetate	-	?
additional information	Thermotoga maritima	the enzyme is active on a variety of acetylated compounds, including cephalosporin C, its esterase activity is confined to short-chain acyl esters of C2-C3 chain length	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Thermotoga maritima	Q9WXT2	gene TM0077	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His-tagged wild-type and selenomethionine-labeled enzymes from Escherichia coli by nickel affinity chromatography, anion exchange chromatography, and gel filtration	Thermotoga maritima

Reaction

Reaction	Comment	Organism	Reaction ID
cephalosporin C + H2O = deacetylcephalosporin C + acetate	a catalytic mechanism in which this Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction	Thermotoga maritima	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
4-nitrophenyl beta-D-xylopyranoside + H2O	4-nitrophenyl-beta-D-xylopyranoside monoacetates as substrates in a beta-xylosidase-coupled assay, TM0077 hydrolyzes acetate at positions 2, 3 and 4 with equal efficiencyas substrates in a ?-xylosidase-coupled assay	Thermotoga maritima	4-nitrophenol + D-xylopyranose	-	?
cephalosporin C + H2O	-	Thermotoga maritima	deacetylcephalosporin C + acetate	-	?
additional information	the enzyme is active on a variety of acetylated compounds, including cephalosporin C, its esterase activity is confined to short-chain acyl esters of C2-C3 chain length	Thermotoga maritima	?	-	?
additional information	the enzyme is an acetyl esterase and not an acetyl xylan esterase, that shows no activity with xylan or acetylated xylan	Thermotoga maritima	?	-	?

Subunits

Subunits	Comment	Organism
hexamer	the relevant physiological oligomeric state of the enzyme is a hexamer, in the crystal structure, two hexamers in the asymmetric unit, that are related by a non-crystallographic two-fold axis, contain each a dimer of trimers with a back-to-back arrangement, enzyme quaternary structure, overview. Two main interfaces play an essential role in complex formation, the first interface between subunit A and B, and he second interface between A and F. The entrance to the internal cavity is blocked by three phenylalanine residues (Phe4), one for each of three monomers that compose half of the hexamer	Thermotoga maritima

Synonyms

Synonyms	Comment	Organism
TM0077	-	Thermotoga maritima

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
100	-	-	Thermotoga maritima

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	-	Thermotoga maritima

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
additional information	-	additional information	inhibition kinetics	Thermotoga maritima

General Information

General Information	Comment	Organism
evolution	the enzyme is a member of the carbohydrate esterase family 7, CE7 family, showing a classic alpha/beta-hydrolase fold	Thermotoga maritima
additional information	the active site has a classic catalytic triad, consisting of Ser188 as the nucleophile, His303 as the proton acceptor/donor, and Asp274 as the acidic residue stabilizing the histidine. The catalytic serine Ser188 is located within a conserved pentapeptide sequence, Gly-X-Ser-X-Gly (GGSQG), characteristic of esterases and lipases, overview	Thermotoga maritima