Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima

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Abstract

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-ß-D-xylopyranoside monoacetates as substrates in a ß-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic a/ß-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction
Original languageEnglish
Pages (from-to)1545-1559
JournalProteins : Structure, Function, and Bioinformatics
Volume80
Issue number6
DOIs
Publication statusPublished - 2012

Fingerprint

Thermotoga maritima
Esterases
Xylans
acetylxylan esterase
Serine
Xylosidases
Phenylmethylsulfonyl Fluoride
Selenomethionine
Paraoxon
Hydrolases
Lipase
Hydroxyl Radical
Conformations
Assays
Tunnels
Esters
Acetates
Carbohydrates
Molecules
Water

Keywords

  • serine-protease mechanism
  • x-ray data
  • crystal-structure
  • xylan-esterase
  • cephalosporin-c
  • macromolecular crystallography
  • substrate-specificity
  • angstrom resolution
  • catalytic serine
  • beta-xylosidase

Cite this

@article{c6dc52212ea846ada3da7f47f4097e30,
title = "Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima",
abstract = "TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-{\ss}-D-xylopyranoside monoacetates as substrates in a {\ss}-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 {\AA} resolution, respectively, revealing a classic a/{\ss}-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 {\AA}, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction",
keywords = "serine-protease mechanism, x-ray data, crystal-structure, xylan-esterase, cephalosporin-c, macromolecular crystallography, substrate-specificity, angstrom resolution, catalytic serine, beta-xylosidase",
author = "M. Levisson and G.W. Han and M.C. Deller and S.N.A. Hendriks and {van der Oost}, J. and S.W.M. Kengen",
year = "2012",
doi = "10.1002/prot.24041",
language = "English",
volume = "80",
pages = "1545--1559",
journal = "Proteins : Structure, Function, and Bioinformatics",
issn = "0887-3585",
publisher = "Wiley",
number = "6",

}

Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima. / Levisson, M.; Han, G.W.; Deller, M.C.; Hendriks, S.N.A.; van der Oost, J.; Kengen, S.W.M.

In: Proteins : Structure, Function, and Bioinformatics, Vol. 80, No. 6, 2012, p. 1545-1559.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima

AU - Levisson, M.

AU - Han, G.W.

AU - Deller, M.C.

AU - Hendriks, S.N.A.

AU - van der Oost, J.

AU - Kengen, S.W.M.

PY - 2012

Y1 - 2012

N2 - TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-ß-D-xylopyranoside monoacetates as substrates in a ß-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic a/ß-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction

AB - TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-ß-D-xylopyranoside monoacetates as substrates in a ß-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic a/ß-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction

KW - serine-protease mechanism

KW - x-ray data

KW - crystal-structure

KW - xylan-esterase

KW - cephalosporin-c

KW - macromolecular crystallography

KW - substrate-specificity

KW - angstrom resolution

KW - catalytic serine

KW - beta-xylosidase

U2 - 10.1002/prot.24041

DO - 10.1002/prot.24041

M3 - Article

VL - 80

SP - 1545

EP - 1559

JO - Proteins : Structure, Function, and Bioinformatics

JF - Proteins : Structure, Function, and Bioinformatics

SN - 0887-3585

IS - 6

ER -