Properties and crystal structure of methylenetetrahydrofolate reductase from Thermus thermophilus HB8.
Properties and crystal structure of methylenetetrahydrofolate reductase from Thermus thermophilus HB8.
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DOI：
10.1371/journal.pone.0023716
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Yamada K
中科院分区：
文献类型：
--
作者：
Igari S;Ohtaki A;Yamanaka Y;Sato Y;Yohda M;Odaka M;Noguchi K;Yamada K
亚甲基四氢叶酸还原酶(MTHFR)是同型半胱氨酸代谢的酶之一。尽管在遗传学和临床上受到了相当大的关注，但由于该酶的生产困难和稳定性差，其反应机制和调控尚不完全清楚。尽管来自嗜热生物的重组酶通常稳定且易于制备，但耐热MTHFRs的性质尚未见报道。嗜热嗜热菌HB8是大肠杆菌MetF的同源物，其亚甲基四氢叶酸还原酶在大肠杆菌中表达和纯化。纯化的MTHFR主要以脱氧亚基和全亚基的杂二聚体形式存在，即每个二聚体结合一个黄素腺嘌呤二核苷酸(FAD)修复基。全亚单位的晶体结构与大肠杆菌MTHFR的β8α8桶非常相似，而非全亚单位的晶体结构是以前没有观察到的封闭形式。此外，晶体中二聚体的亚基间界面不同于大肠杆菌MTHFR四聚体的任何亚基界面。纯化后的Thermus酶可将游离FAD掺入脱辅酶亚基中，形成同源二聚体。通过比较异二聚体和同源二聚体的晶体结构，发现了不同的亚基间界面，表明FAD结合后发生了较大的构象变化。异源二聚体和同源二聚体的大部分生化性质是相同的，除了同源二聚体在叶酸依赖的反应中显示每个Fad结合亚基的≈活性的50%。Thermus MTHFR不同的亚基界面和亚基的重排可能与人类的酶特性有关，如S-腺苷蛋氨酸对变构的调节，以及缺失FAD后Ala222Val突变体的增强不稳定性。虽然大肠杆菌MTHFR是迄今为止人类MTHFR的唯一结构模型，但我们的发现表明Thermus MTHFR将是这种重要酶的另一个有用的模型。
Methylenetetrahydrofolate reductase (MTHFR) is one of the enzymes involved in homocysteine metabolism. Despite considerable genetic and clinical attention, the reaction mechanism and regulation of this enzyme are not fully understood because of difficult production and poor stability. While recombinant enzymes from thermophilic organisms are often stable and easy to prepare, properties of thermostable MTHFRs have not yet been reported. MTHFR from Thermus thermophilus HB8, a homologue of Escherichia coli MetF, has been expressed in E. coli and purified. The purified MTHFR was chiefly obtained as a heterodimer of apo- and holo-subunits, that is, one flavin adenine dinucleotide (FAD) prosthetic group bound per dimer. The crystal structure of the holo-subunit was quite similar to the β8α8 barrel of E. coli MTHFR, while that of the apo-subunit was a previously unobserved closed form. In addition, the intersubunit interface of the dimer in the crystals was different from any of the subunit interfaces of the tetramer of E. coli MTHFR. Free FAD could be incorporated into the apo-subunit of the purified Thermus enzyme after purification, forming a homodimer of holo-subunits. Comparison of the crystal structures of the heterodimer and the homodimer revealed different intersubunit interfaces, indicating a large conformational change upon FAD binding. Most of the biochemical properties of the heterodimer and the homodimer were the same, except that the homodimer showed ≈50% activity per FAD-bound subunit in folate-dependent reactions. The different intersubunit interfaces and rearrangement of subunits of Thermus MTHFR may be related to human enzyme properties, such as the allosteric regulation by S-adenosylmethionine and the enhanced instability of the Ala222Val mutant upon loss of FAD. Whereas E. coli MTHFR was the only structural model for human MTHFR to date, our findings suggest that Thermus MTHFR will be another useful model for this important enzyme.
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影响因子：
120.7
作者：
Graham, IM;Daly, LE;Andria, G
通讯作者：
Andria, G
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.9
作者：
Pejchal, R;Campbell, E;Ludwig, ML
通讯作者：
Ludwig, ML
DOI：
10.1111/j.1432-1033.1990.tb19242.x
发表时间：
1990-09-11
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
WOHLFARTH, G;GEERLIGS, G;DIEKERT, G
通讯作者：
DIEKERT, G
影响因子：
3.2
作者：
Sheppard, CA;Trimmer, EE;Matthews, RG
通讯作者：
Matthews, RG