A phenylalanine clamp controls substrate specificity in the quorum-quenching metallo-γ-lactonase from Bacillus thuringiensis.
A phenylalanine clamp controls substrate specificity in the quorum-quenching metallo-γ-lactonase from Bacillus thuringiensis.
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DOI：
10.1021/bi400050j
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发表时间：
2013-03-05
期刊：
影响因子：
2.9
通讯作者：
Ringe, Dagmar
中科院分区：
文献类型：
--
作者：
Liu, Ce Feng;Liu, Dali;Momb, Jessica;Thomas, Pei W.;Lajoie, Ashley;Petsko, Gregory A.;Fast, Walter;Ringe, Dagmar
AIIA是一种金属依赖的N-酰基高丝氨酸内酯水解酶，具有广泛的底物专一性，但对具有长N-酰基取代的底物表现出偏好。以前，AIIA与开环产物N-己酰-L-高丝氨酸的络合物的晶体结构在金属中心附近显示了结合作用，但没有确定较长底物的N-酰基链的结合口袋。本文报道了一种AIIA突变体F107W的晶体结构，该突变体是在存在和不存在N-癸酰-L-高丝氨酸的情况下测定的。F107位于与先前确定的配体结合口袋相邻的疏水空腔中，而F107W导致与开环产物形成意外的相互作用。值得注意的是，该结构揭示了底物N-酰基链的疏水结合口袋。两个芳香族残基F64和F68形成了一个疏水钳，以产物结合结构的癸酸链中的第七个碳为中心，使得这种相互作用也适用于较长的底物，但不适用于较短的底物。使用不同长度的底物(在疏水钳上带有AIIA突变)的稳态动力学，包括插入氧化还原敏感的半胱氨酸对，证实了这种疏水特征对底物选择性的重要性。确定AIIA的特异性决定因素将有助于开发更具选择性的群体猝灭酶作为工具和潜在的治疗药物。
AiiA is a metal-dependent N-acyl homoserine lactone hydrolase that displays broad substrate specificity, but shows preference for substrates with long N-acyl substitutions. Previously, crystal structures of AiiA in complex with the ring-opened product N-hexanoyl-l-homoserine revealed binding interactions near the metal center, but did not identify a binding pocket for the N-acyl chains of longer substrates. Here we report the crystal structure of an AiiA mutant, F107W, determined in the presence and absence of N-decanoyl-l-homoserine. F107 is located in a hydrophobic cavity adjacent to the previously identified ligand binding pocket, and F107W results in the formation of an unexpected interaction with the ring-opened product. Notably, the structure reveals a previously unidentified hydrophobic binding pocket for the substrate’s N-acyl chain. Two aromatic residues, F64 and F68 form a hydrophobic clamp, centered around the seventh carbon in the product-bound structure’s decanoyl chain, making an interaction that would also be available for longer substrates, but not for shorter substrates. Steady-state kinetics using substrates of various lengths with AiiA bearing mutations at the hydrophobic clamp, including insertion of a redox sensitive cysteine pair, confirms the importance of this hydrophobic feature for substrate preference. Identifying the specificity determinants of AiiA will aid the development of more selective quorum-quenching enzymes as tools and as potential therapeutics.
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影响因子：
2.9
作者：
Momb J;Wang C;Liu D;Thomas PW;Petsko GA;Guo H;Ringe D;Fast W
通讯作者：
Fast W
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：
8
作者：
Huntley, JJA;Fast, W;Dyson, HJ
通讯作者：
Dyson, HJ
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
影响因子：
2.9
作者：
Eyer, P;Worek, F;Reiner, E
通讯作者：
Reiner, E