Structure and catalysis in the Escherichia coli hotdog-fold thioesterase paralogs YdiI and YbdB.
Structure and catalysis in the Escherichia coli hotdog-fold thioesterase paralogs YdiI and YbdB.
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DOI：
10.1021/bi500334v
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发表时间：
2014-07-29
期刊：
影响因子：
2.9
通讯作者：
Dunaway-Mariano D
中科院分区：
文献类型：
--
作者：
Wu R;Latham JA;Chen D;Farelli J;Zhao H;Matthews K;Allen KN;Dunaway-Mariano D
在此，底物识别和催化的结构决定因素在两个热狗折叠硫酯酶旁系同源物，YbdB和YdiI从大肠杆菌，鉴定和分析，以提供深入了解生物功能的进化热狗折叠酶超家族。YbdB和YdiI的X-射线晶体结构，在复杂的惰性底物类似物，在这项研究中确定揭示了各自的硫酯底物结合位点的位置和定位为底物结合和催化的残基的身份。这些残基中的每一个的重要性进行了评估，通过氨基酸置换，然后通过稳态动力学分析相应的定点突变体。瞬态动力学和溶剂18 O-标记的研究，然后进行了深入了解的作用，Glu 63假定作为亲核试剂或一般碱催化。最后，比较了两个旁系同源物的结构-功能-机制概况，沿着与更远的同源物的结构-功能-机制概况，以鉴定底物识别和催化的保守元件，其定义热狗折叠硫酯酶家族的核心性状，以及每个硫酯酶所特有的结构特征。基于从这种分析中获得的洞察力，我们得出结论，在体外底物活性测定揭示的滥交，并假定促进新的生物功能的进化，是产品的内在可塑性在底物结合以及在催化机制。
Herein, the structural determinants for substrate recognition and catalysis in two hotdog-fold thioesterase paralogs, YbdB and YdiI from Escherichia coli, are identified and analyzed to provide insight into the evolution of biological function in the hotdog-fold enzyme superfamily. The X-ray crystal structures of YbdB and YdiI, in complex with inert substrate analogs, determined in this study revealed the locations of the respective thioester substrate binding sites and the identity of the residues positioned for substrate binding and catalysis. The importance of each of these residues was assessed through amino acid replacements followed by steady-state kinetic analyses of the corresponding site-directed mutants. Transient kinetic and solvent 18O-labeling studies were then carried out to provide insight into the role of Glu63 posited to function as the nucleophile or general base in catalysis. Finally, the structure–function–mechanism profiles of the two paralogs, along with that of a more distant homolog, were compared to identify conserved elements of substrate recognition and catalysis, which define the core traits of the hotdog-fold thioesterase family, as well as structural features that are unique to each thioesterase. Founded on the insight gained from this analysis, we conclude that the promiscuity revealed by in vitro substrate activity determinations, and posited to facilitate the evolution of new biological function, is the product of intrinsic plasticity in substrate binding as well as in the catalytic mechanism.
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影响因子：
3.2
作者：
Chen, Minjiao;Ma, Xinyu;Guo, Zhihong
通讯作者：
Guo, Zhihong
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
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作者：
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影响因子：
2.9
作者：
Latham JA;Chen D;Allen KN;Dunaway-Mariano D
通讯作者：
Dunaway-Mariano D
影响因子：
2.9
作者：
Guo, Zu-Feng;Sun, Yueru;Guo, Zhihong
通讯作者：
Guo, Zhihong