Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK.
Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK.
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DOI：
10.1074/jbc.m110.107177
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发表时间：
2010-07-16
期刊：
影响因子：
--
通讯作者：
Blundell TL
中科院分区：
文献类型：
--
作者：
Dias MV;Huang F;Chirgadze DY;Tosin M;Spiteller D;Dry EF;Leadlay PF;Spencer JB;Blundell TL
产氟乙酸链霉菌的硫酯酶FlK催化氟乙酰辅酶A的水解。这提供了一种有效的自我防御机制，防止形成的任何氟乙酰辅酶A被进一步代谢为4-羟基-反式乌头酸，一种三羧酸循环的致命抑制剂。值得注意的是，FlK不接受乙酰辅酶A作为底物。晶体结构分析表明，FlK形成二聚体，其中每个亚基采用热狗折叠，如II型硫酯酶所观察到的。与其他II型硫酯酶，总是利用天冬氨酸或谷氨酸作为催化基地，我们表明，通过定点诱变和晶体学，FlK采用的催化三联体组成的Thr 42，His 76，和水分子，类似于Ser/Cys-His-酸三联体的I型硫酯酶。FlK与各种底物类似物复合的结构比较表明，底物的氟和位于催化三联体对面的Arg 120侧链之间的相互作用对于活性位点处的底物的正确配位是必不可少的，因此占底物特异性。
The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr42, His76, and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg120 located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.