3FSU

Crystal Structure of Escherichia coli Methylenetetrahydrofolate Reductase Double Mutant Phe223LeuGlu28Gln complexed with methyltetrahydrofolate

3FSU の概要

• エントリーDOI: 10.2210/pdb3fsu/pdb
• 関連するPDBエントリー: 3FST
• 分子名称: 5,10-methylenetetrahydrofolate reductase, FLAVIN-ADENINE DINUCLEOTIDE, 5-METHYL-5,6,7,8-TETRAHYDROFOLIC ACID, ... (6 entities in total)
• 機能のキーワード: tim barrel, flavin, reductase, methyltetrahydrofolate, amino-acid biosynthesis, fad, flavoprotein, methionine biosynthesis, nad, nadp, oxidoreductase
• 由来する生物種: Escherichia coli K-12
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 106036.30

構造登録者

Tanner, J.J. (登録日: 2009-01-12, 公開日: 2009-08-25, 最終更新日: 2023-09-06)

主引用文献

Lee, M.N.,Takawira, D.,Nikolova, A.P.,Ballou, D.P.,Furtado, V.C.,Phung, N.L.,Still, B.R.,Thorstad, M.K.,Tanner, J.J.,Trimmer, E.E.
Functional role for the conformationally mobile phenylalanine 223 in the reaction of methylenetetrahydrofolate reductase from Escherichia coli.
Biochemistry, 48:7673-7685, 2009

PubMed Abstract: The flavoprotein methylenetetrahydrofolate reductase from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) by NADH via a ping-pong reaction mechanism. Structures of the reduced enzyme in complex with NADH and of the oxidized Glu28Gln enzyme in complex with CH(3)-H(4)folate [Pejchal, R., Sargeant, R., and Ludwig, M. L. (2005) Biochemistry 44, 11447-11457] have revealed Phe223 as a conformationally mobile active site residue. In the NADH complex, the NADH adopts an unusual hairpin conformation and is wedged between the isoalloxazine ring of the FAD and the side chain of Phe223. In the folate complex, Phe223 swings out from its position in the NADH complex to stack against the p-aminobenzoate ring of the folate. Although Phe223 contacts each substrate in E. coli MTHFR, this residue is not invariant; for example, a leucine occurs at this site in the human enzyme. To examine the role of Phe223 in substrate binding and catalysis, we have constructed mutants Phe223Ala and Phe223Leu. As predicted, our results indicate that Phe223 participates in the binding of both substrates. The Phe223Ala mutation impairs NADH and CH(2)-H(4)folate binding each 40-fold yet slows catalysis of both half-reactions less than 2-fold. Affinity for CH(2)-H(4)folate is unaffected by the Phe223Leu mutation, and the variant catalyzes the oxidative half-reaction 3-fold faster than the wild-type enzyme. Structures of ligand-free Phe223Leu and Phe223Leu/Glu28Gln MTHFR in complex with CH(3)-H(4)folate have been determined at 1.65 and 1.70 A resolution, respectively. The structures show that the folate is bound in a catalytically competent conformation, and Leu223 undergoes a conformational change similar to that observed for Phe223 in the Glu28Gln-CH(3)-H(4)folate structure. Taken together, our results suggest that Leu may be a suitable replacement for Phe223 in the oxidative half-reaction of E. coli MTHFR.

PubMed: 19610625
DOI: 10.1021/bi9007325

実験手法

X-RAY DIFFRACTION (1.7 Å)