Mechanism of substrate specificity in 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidases.
Mechanism of substrate specificity in 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidases.
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DOI：
10.1016/j.jsb.2010.06.006
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发表时间：
2011-01
影响因子：
3
通讯作者：
Howell, P. Lynne
中科院分区：
文献类型：
--
作者：
Siu, Karen K. W.;Asmus, Kyle;Zhang, Allison N.;Horvatin, Cathy;Li, Sheng;Liu, Tong;Moffatt, Barbara;Woods, Virgil L., Jr.;Howell, P. Lynne
关键词：
5′-甲基硫代腺苷/S-腺苷高半胱氨酸（MTA/SAH）核苷酶（MTAN）在细菌和植物的蛋氨酸循环途径中起着关键作用。尽管进行了广泛的结构和生化研究，MTAN底物特异性的分子机制仍然是一个悬而未决的问题。细菌MTAN在水解MTA和SAH方面显示出相当的效率，而植物酶优先选择MTA，对SAH没有活性或活性显著降低。细菌和植物MTAN在整体结构以及腺嘌呤和核糖结合位点上显示出显著的保守性。在拟南芥AtM-TAN 1和AtMTAN 2中发现了一个更为狭窄的5′-烷硫基结合位点，这两个植物MTAN同源物的空间位阻可能在植物MTAN的底物选择中起作用。我们使用等温滴定量热法表明，SAH结合大肠杆菌MTAN（EcMTAN）和AtMTAN 1具有可比的微摩尔亲和力。为了理解为什么AtMTAN 1可以结合但不能水解SAH，我们在2.2 nm分辨率下确定了蛋白质-SAH复合物的结构。缺乏催化活性似乎与酶不能以催化活性方式结合底物有关。动力学在底物选择中的作用也通过探测酰胺质子交换率EcMTAN和AtMTAN 1通过氘-氢交换耦合质谱法进行了检查。这些结果与可用结构的B因子和与底物结合相关的热力学参数相关，并表明EcMTAN活性位点具有更高水平的构象灵活性。我们的研究结果表明，在MTAN中，动力学是底物选择的一个重要因素。
5′-Methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase (MTAN) plays a key role in the methionine-recycling pathway of bacteria and plants. Despite extensive structural and biochemical studies, the molecular mechanism of substrate specificity for MTAN remains an outstanding question. Bacterial MTANs show comparable efficiency in hydrolyzing MTA and SAH, while the plant enzymes select preferentially for MTA, with either no or significantly reduced activity towards SAH. Bacterial and plant MTANs show significant conservation in the overall structure, and the adenine- and ribose-binding sites. The observation of a more constricted 5′-alkylthio binding site in Arabidopsis thaliana AtM-TAN1 and AtMTAN2, two plant MTAN homologues, led to the hypothesis that steric hindrance may play a role in substrate selection in plant MTANs. We show using isothermal titration calorimetry that SAH binds to both Escherichia coli MTAN (EcMTAN) and AtMTAN1 with comparable micromolar affinity. To understand why AtMTAN1 can bind but not hydrolyze SAH, we determined the structure of the protein–SAH complex at 2.2 Å resolution. The lack of catalytic activity appears to be related to the enzyme’s inability to bind the substrate in a catalytically competent manner. The role of dynamics in substrate selection was also examined by probing the amide proton exchange rates of EcMTAN and AtMTAN1 via deuterium–hydrogen exchange coupled mass spectrometry. These results correlate with the B factors of available structures and the thermodynamic parameters associated with substrate binding, and suggest a higher level of conformational flexibility in the active site of EcMTAN. Our results implicate dynamics as an important factor in substrate selection in MTAN.
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影响因子：
5.6
作者：
Hamuro, Y;Burns, LL;Woods, VL
通讯作者：
Woods, VL
影响因子：
5.8
作者：
Gouet, P;Courcelle, E;Métoz, F
通讯作者：
Métoz, F
DOI：
10.1016/j.jasms.2006.06.006
发表时间：
2006-11-01
影响因子：
3.2
作者：
Englander, S. Walter
通讯作者：
Englander, S. Walter
影响因子：
8
作者：
Burns-Hamuro, LL;Hamuro, Y;Woods, VL
通讯作者：
Woods, VL
DOI：
10.1073/pnas.1232301100
发表时间：
2003-06-10
影响因子：
11.1
作者：
Englander, JJ;Del Mar, C;Woods, VL
通讯作者：
Woods, VL