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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- 作者：Robert Pejchal ; Ryan Sargeant ; and Martha L. Ludwig
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：August 30, 2005
- 年：2005
- 卷：44
- 期：34
- 页码：11447 - 11457
- 全文大小：500K
- 年卷期：v.44,no.34(August 30, 2005)
- ISSN：1520-4995
文摘
Methylenetetrahydrofolate reductases (MTHFRs; EC 1.7.99.5) catalyze the NAD(P)H-dependentreduction of 5,10-methylenetetrahydrofolate (CH2-H4folate) to 5-methyltetrahydrofolate (CH3-H4folate)using flavin adenine dinucleotide (FAD) as a cofactor. The initial X-ray structure of Escherichia coliMTHFR revealed that this 33-kDa polypeptide is a (
)8 barrel that aggregates to form an unusual tetramerwith only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Glnenzyme complexed with CH3-H4folate have now been determined at resolutions of 1.95 and 1.85 Å,respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpinconformation and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ringof FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavinring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competentfor hydride transfer. In the complex with CH3-H4folate, the pterin ring is also stacked against FAD in anorientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, asexpected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact withboth folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures revealmultiple conformations for the loops 2-2 (L2), 3-3 (L3), and 4-4 (L4) and suggest that motionsof these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a "closed" conformationthat allows Asp120 to hydrogen bond to the pterin ring in the folate complex but must move to an "open"conformation to allow NADH to bind.
)8 barrel that aggregates to form an unusual tetramerwith only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Glnenzyme complexed with CH3-H4folate have now been determined at resolutions of 1.95 and 1.85 Å,respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpinconformation and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ringof FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavinring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competentfor hydride transfer. In the complex with CH3-H4folate, the pterin ring is also stacked against FAD in anorientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, asexpected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact withboth folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures revealmultiple conformations for the loops 2-2 (L2), 3-3 (L3), and 4-4 (L4) and suggest that motionsof these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a "closed" conformationthat allows Asp120 to hydrogen bond to the pterin ring in the folate complex but must move to an "open"conformation to allow NADH to bind.