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Literature summary 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.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.1.1.6 expressed in Escherichia coli strain DL41 Thermotoga maritima
3.1.1.41 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)

EC Number Crystallization (Comment) Organism
3.1.1.6 hanging drop vapor diffusion method, using 20% (w/v) PEG-3000, 0.1 M HEPES pH 7.5, 0.2 M NaCl (selenomethionine-substituted enzyme) or nanodrop vapor diffusion method, using 0.2 M calcium acetate hydrate, 20% (w/v) PEG 3350, pH 7.3 at 20°C (native enzyme) Thermotoga maritima
3.1.1.41 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

EC Number Inhibitors Comment Organism Structure
3.1.1.6 paraoxon competitive irreversible inhibitor Thermotoga maritima
3.1.1.6 phenylmethylsulfonyl fluoride competitive irreversible inhibitor Thermotoga maritima
3.1.1.41 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
3.1.1.41 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

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.1.1.6 0.137
-
 4-nitrophenyl propionate in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 0.185
-
 4-nitrophenyl acetate in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 3.6
-
 2-O-acetyl-4-nitrophenyl beta-D-xylopyranoside in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 4
-
 4-O-acetyl-4-nitrophenyl beta-D-xylopyranoside in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 4.2
-
 3-O-acetyl-4-nitrophenyl beta-D-xylopyranoside in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima

Localization

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

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.1.1.41 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
3.1.1.41 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)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.1.1.41 cephalosporin C + H2O Thermotoga maritima
-
 deacetylcephalosporin C + acetate
-
 ?
3.1.1.41 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

EC Number Organism UniProt Comment Textmining
3.1.1.6 Thermotoga maritima
-
-
-
3.1.1.41 Thermotoga maritima Q9WXT2 gene TM0077
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.1.1.6 nickel-chelating resin column chromatography and Superdex 200 gel filtration Thermotoga maritima
3.1.1.41 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

EC Number Reaction Comment Organism Reaction ID
3.1.1.41 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.1.1.6 1-naphthyl acetate + H2O
-
 Thermotoga maritima 1-naphthol + acetate
-
 ?
3.1.1.6 2-O-acetyl-4-nitrophenyl beta-D-xylopyranoside + H2O
-
 Thermotoga maritima ?
-
 ?
3.1.1.6 3-O-acetyl-4-nitrophenyl beta-D-xylopyranoside + H2O
-
 Thermotoga maritima ?
-
 ?
3.1.1.6 4-nitrophenyl acetate + H2O
-
 Thermotoga maritima 4-nitrophenol + acetate
-
 ?
3.1.1.6 4-nitrophenyl propionate + H2O
-
 Thermotoga maritima 4-nitrophenol + propionate
-
 ?
3.1.1.6 4-O-acetyl-4-nitrophenyl beta-D-xylopyranoside + H2O
-
 Thermotoga maritima ?
-
 ?
3.1.1.6 7-aminocephalosporanic acid + H2O
-
 Thermotoga maritima ?
-
 ?
3.1.1.6 cephalosporin C + H2O
-
 Thermotoga maritima deacetylcephalosporin C + acetate
-
 ?
3.1.1.6 glucose penta-acetate + H2O high activity Thermotoga maritima ?
-
 ?
3.1.1.6 additional information no activity is detected on xylan or acetylated xylan or N-acetyl-D-glucosamine Thermotoga maritima ?
-
 ?
3.1.1.41 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
-
 ?
3.1.1.41 cephalosporin C + H2O
-
 Thermotoga maritima deacetylcephalosporin C + acetate
-
 ?
3.1.1.41 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 ?
-
 ?
3.1.1.41 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

EC Number Subunits Comment Organism
3.1.1.41 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

EC Number Synonyms Comment Organism
3.1.1.6 Acetyl esterase
-
 Thermotoga maritima
3.1.1.6 TM0077
-
 Thermotoga maritima
3.1.1.41 TM0077
-
 Thermotoga maritima

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.1.1.6 100
-
-
 Thermotoga maritima
3.1.1.41 100
-
-
 Thermotoga maritima

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.1.1.6 100
-
 the enzyme is not stable at 100°C, resulting in a half-life of less than 5 min Thermotoga maritima

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.1.1.6 41.3
-
 4-nitrophenyl propionate in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 57.5
-
 4-nitrophenyl acetate in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 70.1
-
 3-O-acetyl-4-nitrophenyl beta-D-xylopyranoside in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 76.1
-
 2-O-acetyl-4-nitrophenyl beta-D-xylopyranoside in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 78.6
-
 4-O-acetyl-4-nitrophenyl beta-D-xylopyranoside in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.1.1.6 7.5
-
-
 Thermotoga maritima
3.1.1.41 7.5
-
-
 Thermotoga maritima

Ki Value [mM]

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

General Information

EC Number General Information Comment Organism
3.1.1.41 evolution the enzyme is a member of the carbohydrate esterase family 7, CE7 family, showing a classic alpha/beta-hydrolase fold Thermotoga maritima
3.1.1.41 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

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.1.1.6 16.7
-
 3-O-acetyl-4-nitrophenyl beta-D-xylopyranoside in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 19.7
-
 4-O-acetyl-4-nitrophenyl beta-D-xylopyranoside in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 21.1
-
 2-O-acetyl-4-nitrophenyl beta-D-xylopyranoside in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 301.5
-
 4-nitrophenyl propionate in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima
3.1.1.6 310.8
-
 4-nitrophenyl acetate in 50 mM citrate-phosphate (pH 6) at 70°C Thermotoga maritima