Catalytic Mechanism of the Hotdog-Fold Thioesterase PA1618 Revealed by X-ray Structure Determination of a Substrate-Bound Oxygen Ester Analogue Complex.
Catalytic Mechanism of the Hotdog-Fold Thioesterase PA1618 Revealed by X-ray Structure Determination of a Substrate-Bound Oxygen Ester Analogue Complex.
复制标题
通过底物结合氧酯类似物复合物的 X 射线结构测定揭示热狗折叠硫酯酶 PA1618 的催化机制。
DOI：
10.1002/cbic.201700322
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Dunaway-Mariano,Debra
中科院分区：
文献类型：
--
作者：
Latham,JohnA;Ji,Tianyang;Matthews,Kaila;Mariano,PatrickS;Allen,KarenN;Dunaway-Mariano,Debra
在热狗折叠超家族中，硫代酯酶活性占大部分活性。许多热狗酶的催化结构和机理已经通过X射线结晶学和动力学来阐明，以探索特定的底物使用和细胞功能。然而，迄今为止报道的热狗硫代酯酶与底物类似物的复合体结构利用包含底物截断或包括额外原子的配体来防止水解。在这里，我们合成了一种等空间和等电子的底物类似物苯甲酰-OdCoA，并研究了它与铜绿假单胞菌热狗硫代酯酶的配合物PA1618的X射线晶体结构(1.72？分辨率)。将该络合物与“不完美”的底物类似物苯乙酰-辅酶A进行比较，后者的分辨率提高到1.62？动力学和结构结果与Glu64作为催化残基和Gln49稳定过渡态是一致的。对两种配体结合结构的结构比较发现，在苯甲酰基-OdCoA结构的活性中心配位有一个关键的有序水分子，而苯酰基-CoA结合结构中没有这种结构。这表明了一种通用的碱基催化机制，即Glu64激活配位的水亲核试剂。总之，我们的发现揭示了更难区分的底物类似物对于确定适当的底物结合和催化机制的重要性。
Thioesterase activity accounts for the majority of the activities in the hotdog-fold superfamily. The structure and mechanism of catalysis for many hotdog enzymes have been elucidated by X-ray crystallography and kinetics to probe the specific substrate usage and cellular functions. However, structures of hotdog thioesterases in complex with substrate analogs reported to date utilize ligands that comprise either truncations of the substrate or include additional atoms to prevent the hydrolysis. Herein, we present the synthesis of an isosteric and isoelectronic substrate analog, benzoyl-OdCoA, and the X-ray crystal structure of a complex of the analog with P. aeruginosa hotdog thioesterase, PA1618 (at 1.72 Å resolution). The complex is compared to that of the “imperfect” substrate analog phenacyl-CoA, refined to a resolution of 1.62 Å. Kinetic and structural results are consistent with Glu64 as the catalytic residue and with Gln49 in stabilization of the transition state. Structural comparison of the two ligand-bound structures revealed a crucial ordered water molecule coordinated in the active site of the benzoyl-OdCoA structure which is not present in the phenacyl-CoA bound structure. This suggests a general base mechanism of catalysis where Glu64 activates the coordinated water nucleophile. Together, our findings reveal the importance of a more indistinguishable substrate analog to determine proper substrate binding and catalytic mechanism.