Structures of NADH and CH3-H4folate complexes of Escherichia coli methylenetetrahydrofolate reductase reveal a Spartan strategy for a ping-pong reaction
Structures of NADH and CH3-H4folate complexes of Escherichia coli methylenetetrahydrofolate reductase reveal a Spartan strategy for a ping-pong reaction
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DOI：
10.1021/bi050533q
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发表时间：
2005-08-30
期刊：
影响因子：
2.9
通讯作者：
Ludwig, ML
中科院分区：
文献类型：
--
作者：
Pejchal, R;Sargeant, R;Ludwig, ML
亚甲基四氢叶酸还原酶(MTHFRs；EC 1.7.99.5)以黄素腺嘌呤二核苷酸(FAD)为辅因子，催化NAD(P)依赖的5，10-亚甲基四氢叶酸(CH2-H(4)叶酸)还原为5-甲基四氢叶酸(CH3-H(4)叶酸)。大肠杆菌MTHFR的初步X射线结构表明，这个33 kDa的多肽是一个(βα)8桶，聚集形成一个不寻常的四聚体，只有2重对称性。还原酶与NADH络合的结构和氧化型Glu28Gln酶与CH3-H(4)叶酸络合的结构分别以1.95埃和1.85埃的分辨率被测定。NADH复合体揭示了一种罕见的二核苷酸结合模式；NADH采用发夹构象，并夹在保守的苯丙氨酸Phe223和FAD的异异丙嗪环之间。结合的吡啶核苷酸的烟酰胺与FAD的N5相邻的C4与黄素环的表面堆积在一起，这意味着该结构模拟了一种能够进行氢化物转移的络合物。在具有CH3-H4叶酸的络合物中，蝶呤环也以有利于氢化物转移的方向堆积在FAD上。因此，正如许多催化乒乓球反应的酶所预期的那样，两种底物的结合部位重叠，并且几个不变残基与叶酸和吡啶核苷酸底物相互作用。通过比较配体结构和无底物结构，揭示了β2-α2(L2)、β3-α3(L3)和β4-α4(1-4)环的多种构象，并表明这些环的运动促进了乒乓球反应。尤其是，L4环采用一种“封闭”构象，允许Asp 120与叶酸复合体中的蝶呤环氢键结合，但必须移动到“开放”构象才能与NADH结合。
Methylenetetrahydrofolate reductases (MTHFRs; EC 1.7.99.5) catalyze the NAD(P)H-dependent reduction of 5,10-methylenetetrahydrofolate (CH2-H(4)folate) to 5-methyltetrahydrofolate (CH3-H(4)folate) using flavin adenine dinucleotide (FAD) as a cofactor. The initial X-ray structure of Escherichia coli MTHFR revealed that this 33-kDa polypeptide is a (beta alpha)8 barrel that aggregates to form an unusual tetramer with only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Gln enzyme complexed with CH3-H(4)folate have now been determined at resolutions of 1.95 and 1.85 angstrom, respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpin conformation and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ring of FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavin ring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competent for hydride transfer. In the complex with CH3-H4folate, the pterin ring is also stacked against FAD in an orientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, as expected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact with both folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures reveal multiple conformations for the loops beta 2-alpha 2 (L2), beta 3-alpha 3 (L3), and beta 4-alpha 4 (1-4) and suggest that motions of these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a "closed" conformation that allows Asp 120 to hydrogen bond to the pterin ring in the folate complex but must move to an "open" conformation to allow NADH to bind.