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Functional role for the conformationally mobile phenylalanine 223 in the reaction of methylenetetrahydrofolate reductase from Escherichia coli.

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.

SUBMITTER: Lee MN

PROVIDER: S-EPMC2805098 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Functional role for the conformationally mobile phenylalanine 223 in the reaction of methylenetetrahydrofolate reductase from Escherichia coli.

Lee Moon N MN Takawira Desire D Nikolova Andriana P AP Ballou David P DP Furtado Vivek C VC Phung Ngoc L NL Still Brady R BR Thorstad Melissa K MK Tanner John J JJ Trimmer Elizabeth E EE

Biochemistry 20090801 32

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 NA ...[more]

PMID: 19610625

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Project description:BackgroundThe rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed.ResultsMTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway.ConclusionThe identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains.

Dataset Information

Functional role for the conformationally mobile phenylalanine 223 in the reaction of methylenetetrahydrofolate reductase from Escherichia coli.

Publications

Functional role for the conformationally mobile phenylalanine 223 in the reaction of methylenetetrahydrofolate reductase from Escherichia coli.

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