Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima.
Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima.
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DOI：
10.1002/prot.24041
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发表时间：
2012-06
影响因子：
2.9
通讯作者：
Wilson, Ian A.
中科院分区：
文献类型：
--
作者：
Levisson, Mark;Han, Gye Won;Deller, Marc C.;Xu, Qingping;Biely, Peter;Hendriks, Sjon;Ten Eyck, Lynn F.;Flensburg, Claus;Roversi, Pietro;Miller, Mitchell D.;McMullan, Daniel;von Delft, Frank;Kreusch, Andreas;Deacon, Ashley M.;van der Oost, John;Lesley, Scott A.;Elsliger, Marc-Andre;Kengen, Serve W. M.;Wilson, Ian A.
来自海栖热袍菌的TM 0077是碳水化合物酯酶家族7的成员，对多种乙酰化化合物具有活性，包括头孢菌素C。TM 0077酯酶活性仅限于短链酰基酯（C2-C3），在100°C和pH 7.5左右最佳。在β-木糖苷酶偶联试验中，使用4-硝基苯基-β-D-吡喃木糖苷单乙酸酯作为底物，研究了TM 0077的位置特异性。TM 0077在位置2、3和4以相同的效率水解乙酸盐。在木聚糖或乙酰化木聚糖上未检测到活性，这意味着TM 0077是乙酰酯酶，而不是目前注释的乙酰木聚糖酯酶。分别在2.1 nm和2.5 nm分辨率下测定了TM 0077的硒代蛋氨酸取代和天然结构，揭示了经典的α/β-水解酶折叠。TM 0077组装成一个甜甜圈状的六聚体，两侧有小隧道通向内腔，其中包含六个催化中心。与苯甲基磺酰氟（PMSF）和对氧磷共价结合的TM 0077的结构分别测定为2.4 μ m和2.1 μ m，并证实两种抑制剂均与催化丝氨酸（Ser 188）共价结合。抑制剂结合后，催化丝氨酸采用改变的构象，如在其他酯酶和脂肪酶中观察到的，并支持先前提出的催化机制，其中该丝氨酸羟基旋转防止反应逆转，并允许水分子进入以完成反应。
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-β-D-xylopyranoside monoacetates as substrates in a β-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 Å and 2.5 Å resolution, respectively, revealing a classic α/β-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 (PMSF) and paraoxon were determined to 2.4 Å 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 this Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction.
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影响因子：
2.9
作者：
Febbraio, Ferdinando;D'Andrea, Sandro Esposito;Manco, Giuseppe
通讯作者：
Manco, Giuseppe
影响因子：
5.1
作者：
BIZZOZERO, SA;DUTLER, H
通讯作者：
DUTLER, H
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.2
作者：
Conners, SB;Montero, CI;Kelly, RM
通讯作者：
Kelly, RM
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH