Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Record number 427457
Title Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima
Author(s) Levisson, M.; Han, G.W.; Deller, M.C.; Hendriks, S.N.A.; Oost, J. van der; Kengen, S.W.M.
Source Proteins : Structure, Function, and Bioinformatics 80 (2012)6. - ISSN 0887-3585 - p. 1545 - 1559.
DOI https://doi.org/10.1002/prot.24041
Department(s) Microbiological Laboratory
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2012
Keyword(s) serine-protease mechanism - x-ray data - crystal-structure - xylan-esterase - cephalosporin-c - macromolecular crystallography - substrate-specificity - angstrom resolution - catalytic serine - beta-xylosidase
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
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