The crystal structure of tetrameric methionine adenosyltransferase from rat liver reveals the methionine-binding site
The crystal structure of tetrameric methionine adenosyltransferase from rat liver reveals the methionine-binding site
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DOI：
10.1006/jmbi.2000.3858
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发表时间：
2000-07-07
影响因子：
5.6
通讯作者：
Sanz-Aparicio, J
中科院分区：
文献类型：
--
作者：
González, B;Pajares, MA;Sanz-Aparicio, J
大多数转甲基化反应使用相同的甲基供体，s -腺苷蛋氨酸（SAM），由蛋氨酸和ATP通过蛋氨酸腺苷转移酶（MAT）合成。在哺乳动物中，已经检测到两种MAT酶，一种普遍存在，另一种是肝脏特异性的。肝酶以两种低聚形式存在，四聚体（MAT I）和二聚体（MAT III），其中MAT I对蛋氨酸的亲和力较高，但合成SAM的能力较低。我们在2.7埃的分辨率下解决了大鼠肝脏MAT I的晶体结构，并与蛋氨酸类似物络合：l -2-氨基-4-甲氧基顺式-3-烯酸（L-cisAMB）。该酶由四个相同的亚基组成，排列在两个紧密的二聚体中，这些二聚体通过晶体学上的2倍对称联系在一起。晶体结构显示了相关半胱氨酸残基在链上的位置，并且Cys35和Cys61完全定向形成二硫链。这一结果使我们提出了一个假设来解释MAT I/III交换的控制，从而解释对活性的影响。我们首次在活性位点空腔中发现了蛋氨酸结合位点。L-cisAMB抑制剂以相当平面的构象堆叠在Phe251芳香环上，其羧酸基配位为Mg2+，而Mg2+又与Asp180相连。相关残基在MAT活性中的重要作用已被定点诱变证实。Phe251暴露于溶剂中，位于连接n端结构域和中心结构域的柔性环Phe251- ala260的开始。我们假设在酶促反应期间可能发生构象变化，这可能是涉及三聚磷酸盐水解的不寻常的两步机制的原因。根据研究结果，讨论了其他重要的力学意义。此外，本研究中确定的某些残基在蛋氨酸识别中可能具有的关键作用为合理的药物设计提供了进一步的可能性。(C) 2000年学术出版社。
Most of the transmethylation reactions use the same methyl donor, S-adenosylmethionine (SAM), that is synthesised from methionine and ATP by methionine adenosyltransferase (MAT). In mammals, two MAT enzymes have been detected, one ubiquitous and another liver specific. The liver enzyme exists in two oligomeric forms, a tetramer (MAT I) and a dimer (MAT III), MAT I being the one that shows a higher level of affinity for methionine but a lower SAM synthesis capacity. We have solved the crystal structure of rat liver MAT I at 2.7 Angstrom resolution, complexed with a methionine analogue: L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB). The enzyme consists of four identical subunits arranged in two tight dimers that are related by crystallographic 2-fold symmetry. The crystal structure shows the positions of the relevant cysteine residues in the chain, and that Cys35 and Cys61 are perfectly oriented for forming a disulphide link. This result leads us to propose a hypothesis to explain the control of MAT I/III exchange and hence, the effects observed on activity. We have identified the methionine-binding site into the active-site cavity, for the first time. The L-cisAMB inhibitor is stacked against Phe251 aromatic ring in a rather planar conformation, and its carboxylate group coordinates a Mg2+, which, in turn, is linked to Asp180. The essential role of the involved residues in MAT activity has been confirmed by site-directed mutagenesis. Phe251 is exposed to solvent and is located in the beginning of the flexible loop Phe251-Ala260 that is connecting the N-terminal domain to the central domain. We postulate that a conformational change may take place during the enzymatic reaction and this is possibly the reason of the unusual two-step mechanism involving tripolyphosphate hydrolysis. Other important mechanistic implications are discussed on the light of the results. Moreover, the critical role that certain residues identified in this study may have in methionine recognition opens further possibilities for rational drug design. (C) 2000 Academic Press.