Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryoelectron microscopy.
Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryoelectron microscopy.
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DOI：
10.1073/pnas.2302531120
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发表时间：
2023-06-27
影响因子：
11.1
通讯作者：
Ando, Nozomi
中科院分区：
文献类型：
--
作者：
Watkins, Maxwell B.;Wang, Haoyue;Burnim, Audrey;Ando, Nozomi
甲硫氨酸合成酶利用维生素B12和B 9的衍生物在生物甲基化化学中发挥重要作用。甲硫氨酸合酶的缺陷与人类发育障碍和其他健康后果有关。数十年来对蛋氨酸合成酶的生化和结构研究表明，这种酶必须是高度动态的，在三个不同的活性位点之间移动其辅因子。在这项研究中，我们使用先进的结构方法和结构预测相结合，提供一个整体的看法全长蛋氨酸合酶，并描述这种灵活的酶如何启动催化和切换到其再激活模式时，酶变得氧化失活。钴胺素依赖性甲硫氨酸合酶（MetH）利用其辅因子的独特化学作用催化同型半胱氨酸和5-甲基四氢叶酸（CH 3-H4叶酸）合成甲硫氨酸。在这样做时，MetH将S-腺苷甲硫氨酸的循环与一碳代谢中的叶酸循环联系起来。对大肠杆菌MetH的广泛的生物化学和结构研究表明，这种灵活的多结构域酶采用两种主要构象来防止蛋氨酸生产和消耗的无效循环。然而，由于MetH是高度动态的，并且既是光敏的又是氧敏感的金属酶，它对结构研究提出了特殊的挑战，现有的结构必然来自“分而治之”的方法。在这项研究中，我们调查了E。使用小角X射线散射（SAXS）、单粒子冷冻电子显微镜（cryo-EM）以及对AlphaFold 2数据库的广泛分析，对大肠杆菌MetH和丝状栖热菌的嗜热同源物进行了研究，以完整地呈现全长MetH的结构描述。使用SAXS，我们描述了一个共同的静息态构象共享的活性和非活性氧化态的MetH和CH 3-H4叶酸和flavodoxin在启动营业额和再激活的作用。通过结合小角X射线散射和T. filiformis MetH，我们表明，静息态构象由一个稳定的催化结构域，连接到一个高度移动的再活化结构域的安排。最后，通过结合AlphaFold 2引导的序列分析和我们的实验结果，我们提出了一个通用模型的功能开关MetH。
The enzyme methionine synthase utilizes derivatives of vitamins B12 and B9 to play an essential role in biological methylation chemistry. Deficiencies in methionine synthase have been linked to developmental disorders and other health consequences in humans. Decades of biochemical and structural work on methionine synthase have shown that this enzyme must be highly dynamic, moving its cofactor between three different active sites. In this study, we use a combination of advanced structural methods and structure prediction to provide a wholistic view of the full-length methionine synthase and describe how this flexible enzyme initiates catalysis and switches to its reactivation mode when the enzyme becomes oxidatively inactivated. Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH3-H4folate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling of S-adenosylmethionine with the folate cycle in one-carbon metabolism. Extensive biochemical and structural studies on Escherichia coli MetH have shown that this flexible, multidomain enzyme adopts two major conformations to prevent a futile cycle of methionine production and consumption. However, as MetH is highly dynamic as well as both a photosensitive and oxygen-sensitive metalloenzyme, it poses special challenges for structural studies, and existing structures have necessarily come from a “divide and conquer” approach. In this study, we investigate E. coli MetH and a thermophilic homolog from Thermus filiformis using small-angle X-ray scattering (SAXS), single-particle cryoelectron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present a structural description of the full-length MetH in its entirety. Using SAXS, we describe a common resting-state conformation shared by both active and inactive oxidation states of MetH and the roles of CH3-H4folate and flavodoxin in initiating turnover and reactivation. By combining SAXS with a 3.6-Å cryo-EM structure of the T. filiformis MetH, we show that the resting-state conformation consists of a stable arrangement of the catalytic domains that is linked to a highly mobile reactivation domain. Finally, by combining AlphaFold2-guided sequence analysis and our experimental findings, we propose a general model for functional switching in MetH.
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影响因子：
56.9
作者：
DRENNAN, CL;HUANG, S;LUDWIG, ML
通讯作者：
LUDWIG, ML
影响因子：
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2.9
作者：
BANERJEE, RV;FRASCA, V;MATTHEWS, RG
通讯作者：
MATTHEWS, RG
影响因子：
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作者：
Huang, Sha;Romanchuk, Gail;Ludwig, Martha
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影响因子：
2.9
作者：
Jarrett, JT;Hoover, DM;Matthews, RG
通讯作者：
Matthews, RG