Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography.
Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography.
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DOI：
10.1038/s42004-023-00964-9
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发表时间：
2023-08-03
影响因子：
5.9
通讯作者：
Kovalevsky, Andrey
中科院分区：
文献类型：
--
作者：
Drago, Victoria N. N.;Campos, Claudia;Hooper, Mattea;Collins, Aliyah;Gerlits, Oksana;Weiss, Kevin L. L.;Blakeley, Matthew P. P.;Phillips, Robert S. S.;Kovalevsky, Andrey
吡哆醛 5'-磷酸 (PLP) 依赖性酶利用维生素 B6 衍生的辅因子对氨基酸和其他小分子进行大量化学转化。一些 PLP 依赖性酶，例如丝氨酸羟甲基转移酶 (SHMT)，是设计小分子抗菌药物和抗癌疗法的有希望的药物靶标，而其他酶则已用于合成药物构件。了解 PLP 依赖性催化和反应特异性对于推进结构辅助药物设计和酶工程至关重要。在这里，我们报告了使用室温联合 X 射线/中子晶体学直接测定内部醛亚胺状态的嗜热栖热菌 SHMT (TthSHMT) 活性位点的质子化态。通过获得 hSHMT2 的室温 X 射线结构，比较模型酶 TthSHMT 和人线粒体 SHMT (hSHMT2) 的保守活性位点结构，表明人酶中具有相同的质子化状态。跟踪通过 TthSHMT 活性位点腔的氨基酸底物丝氨酸途径，揭示了分别对应于前米氏复合物和假米氏复合物的外周和阳离子结合位点。在外围结合位点，底物以两性离子形式结合。通过分析观察到的质子化状态，建议使用 Glu53（而非 His 残基）作为通用碱催化剂，协调 L-丝氨酸逆羟醛转化为甘氨酸。丝氨酸羟甲基转移酶 (SHMT) 是一种吡哆醛 5'-磷酸 (PLP) 依赖性酶，也是一个有前途的药物靶点，然而，PLP 依赖性催化作用仍未得到充分探索。在这里，作者报告了嗜热栖热菌 SHMT 在内部醛亚胺状态下以及与外围结合位点处的 L-丝氨酸底物形成的复合物的联合 X 射线/中子结构，揭示了氢原子的位置，确定了残基的质子化状态和电荷，并提出了催化机制。
Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B6-derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine. Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme and a promising drug target, however, PLP-dependent catalysis remains underexplored. Here, the authors report joint X-ray/neutron structures of Thermus thermophilus SHMT in the internal aldimine state and in complex with L-serine substrate trapped at the peripheral binding site, revealing positions of hydrogen atoms, assigning the protonation states and electrical charges of residues, and proposing a catalytic mechanism.
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影响因子：
2.7
作者：
Cui X;Cui Y;Du T;Jiang X;Song C;Zhang S;Ma C;Liu Y;Ni Q;Gao Y;Wang G
通讯作者：
Wang G
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DOI：
10.1107/s0907444909011548
发表时间：
2009-06
影响因子：
2.2
作者：
Adams, Paul D.;Mustyakimov, Marat;Afonine, Pavel V.;Langan, Paul
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Langan, Paul
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作者：
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Munoz, Francisco
影响因子：
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影响因子：
5.8
作者：
Ota T;Senoo A;Shirakawa M;Nonaka H;Saito Y;Ito S;Ueno G;Nagatoishi S;Tsumoto K;Sando S
通讯作者：
Sando S