Folate Activation and Catalysis in Methylenetetrahydrofolate Reductase from Escherichia coli: Roles for Aspartate 120 and Glutamate 28

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• 作者：Elizabeth E. Trimmer ; David P. Ballou ; Martha L. Ludwig ; and Rowena G. Matthews
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：May 29, 2001
• 年：2001
• 卷：40
• 期：21
• 页码：6216 - 6226
• 全文大小：156K
• 年卷期：v.40,no.21(May 29, 2001)
• ISSN：1520-4995

文摘

The flavoprotein Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzesthe reduction of 5,10-methylenetetrahydrofolate (CH₂-H₄folate) to 5-methyltetrahydrofolate (CH₃-H₄folate).The X-ray crystal structure of the enzyme has revealed the amino acids at the flavin active site that arelikely to be relevant to catalysis. Here, we have focused on two conserved residues, Asp 120 and Glu 28.The presence of an acidic residue (Asp 120) near the N1-C2=O position of the flavin distinguishesMTHFR from all other known flavin oxidoreductases and suggests an important function for this residuein modulating the flavin reactivity. Modeling of the CH₃-H₄folate product into the enzyme active sitealso suggests roles for Asp 120 in binding of folate and in electrostatic stabilization of the putative 5-iminiumcation intermediate during catalysis. In the NADH-menadione oxidoreductase assay and in the isolatedreductive half-reaction, the Asp120Asn mutant enzyme is reduced by NADH 30% more rapidly than thewild-type enzyme, which is consistent with a measured increase in the flavin midpoint potential. Comparedto the wild-type enzyme, the mutant showed 150-fold decreased activity in the physiological NADH-CH₂-H₄folate oxidoreductase reaction and in the oxidative half-reaction involving CH₂-H₄folate, but theapparent K_d for CH₂-H₄folate was relatively unchanged. Our results support a role for Asp 120 in catalysisof folate reduction and perhaps in stabilization of the 5-iminium cation. By analogy to thymidylate synthase,which also uses CH₂-H₄folate as a substrate, Glu 28 may serve directly or via water as a general acidcatalyst to aid in 5-iminium cation formation. Consistent with this role, the Glu28Gln mutant was unableto catalyze the reduction of CH₂-H₄folate and was inactive in the physiological oxidoreductase reaction.The mutant enzyme was able to bind CH₃-H₄folate, but reduction of the FAD cofactor was not observed.In the NADH-menadione oxidoreductase assay, the mutant demonstrated a 240-fold decrease in activity.