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Escherichia coli K-12 substr. MG1655 Enzyme: 5,10-methylenetetrahydrofolate reductase



Gene: metF Accession Numbers: EG10585 (EcoCyc), b3941, ECK3933

Regulation Summary Diagram: ?

Subunit composition of 5,10-methylenetetrahydrofolate reductase = [MetF]4
         5,10-methylenetetrahydrofolate reductase = MetF

Summary:
E. coli MetF is a flavoprotein capable of catalyzing the NADH-linked reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. The enzyme is a tetramer of four identical subunits with a beta8alpha8 barrel catalytic domain. Each of the four subunits of MetH contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD) at the C-termini of the beta strands [Donaldson62, Larrabee63, Cathou63, Guest64a, Katzen65, SaintGirons83].

metJ mutants show that MetJ proteins regulate the vitamin B12-mediated MetF expression [Belfaiza87, Cai92].

The mutation A177V near the catalytic domain of MetF increases the chance to lose its essential flavin cofactor [Guenther99]. Flavin dissociation is associated with the loss of enzymatic activity. The native tetramer of the enzyme dissociates into dimers preceding flavin release at low enzyme concentration. An enzymatically active holodimer of MetF is the smallest functionally active unit of the enzyme because the FAD dissociation occurs only on unfolding of the holodimer to monomer [Misra03].

Gene Citations: [Greene84]

Locations: cytosol

Map Position: [4,130,639 -> 4,131,529] (89.03 centisomes)
Length: 891 bp / 296 aa

Molecular Weight of Polypeptide: 33.103 kD (from nucleotide sequence)

pI: 6.39

Unification Links: ASAP:ABE-0012897 , CGSC:511 , EchoBASE:EB0580 , EcoGene:EG10585 , EcoliWiki:b3941 , Entrez-gene:948432 , ModBase:P0AEZ1 , OU-Microarray:b3941 , PortEco:metF , PR:PRO_000023212 , Pride:P0AEZ1 , Protein Model Portal:P0AEZ1 , RefSeq:NP_418376 , RegulonDB:EG10585 , SMR:P0AEZ1 , String:511145.b3941 , Swiss-Model:P0AEZ1 , UniProt:P0AEZ1

Relationship Links: InterPro:IN-FAMILY:IPR003171 , InterPro:IN-FAMILY:IPR004620 , PDB:Structure:1B5T , PDB:Structure:1ZP3 , PDB:Structure:1ZP4 , PDB:Structure:1ZPT , PDB:Structure:1ZRQ , PDB:Structure:2FMN , PDB:Structure:2FMO , PDB:Structure:3FST , PDB:Structure:3FSU , Pfam:IN-FAMILY:PF02219

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0046654 - tetrahydrofolate biosynthetic process Inferred from experiment [Sheppard99]
GO:0051289 - protein homotetramerization Inferred from experiment [Sheppard99]
GO:0006555 - methionine metabolic process Inferred by computational analysis [GOA01]
GO:0008652 - cellular amino acid biosynthetic process Inferred by computational analysis [UniProtGOA11]
GO:0009086 - methionine biosynthetic process Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0035999 - tetrahydrofolate interconversion Inferred by computational analysis [UniProtGOA12]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11, GOA01]
Molecular Function: GO:0004489 - methylenetetrahydrofolate reductase (NAD(P)H) activity Inferred from experiment Inferred by computational analysis [GOA01a, GOA01, Sheppard99]
GO:0071949 - FAD binding Inferred from experiment [Sheppard99]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11]
GO:0051087 - chaperone binding [Kerner05]
Cellular Component: GO:0005829 - cytosol Inferred by computational analysis [GOA01, DiazMejia09]

MultiFun Terms: metabolism central intermediary metabolism formyl-THF biosynthesis

Essentiality data for metF knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 0.4% glucose No 37 Aerobic 7.2 0.27 No [Patrick07, Comment 3]
M9 medium with 1% glycerol No 37 Aerobic 7.2 0.35 No [Joyce06]
MOPS medium with 0.4% glucose Indeterminate 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
No [Feist07, Comment 4]

Credits:
Reviewed 01-Mar-2010 by Sarker M


Enzymatic reaction of: 5,10-methylenetetrahydrofolate reductase

Synonyms: 5-methyltetrahydrofolate:(acceptor) oxidoreductase

EC Number: 1.5.1.20

an N5-methyl-tetrahydrofolate + NAD+ <=> a 5,10-methylene-tetrahydrofolate + NADH + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

This reaction is reversible.

Alternative Substrates for NADH: NADPH [Sheppard99 ]

In Pathways: N10-formyl-tetrahydrofolate biosynthesis

Summary:
E. coli MetF is a flavoprotein capable of catalyzing the NADH-linked reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Each of the four subunits of MetH contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). In the reaction, NADH transfers reducing equivalents to the FAD cofactor, which in turn transfers them to methyltetrahydrofolate. This reaction provides methyltetrahydrofolate to be used for methylation of homocysteine to form methionine [Neidhardt96]. NADPH also serves as a reductant, but much less effectively than NADH. MetF can also catalyze the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an artificial electron acceptor [Sheppard99].

The mutation A177V near the catalytic domain of MetF increases the chance to lose its essential flavin cofactor [Guenther99].

The enzyme catalyzes these oxidoreductions by a ping-pong Bi-Bi mechanism. Reoxidation of the reduced flavin by methylenetetrahydrofolate is rate limiting in the physiological NADH-methylenetetrahydrofolate oxidoreductase reaction. In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting [Trimmer01].

Studies with Asp120Asn and Glu28Gln mutant enzymes support the involvement of conserved acid residues Asp120 and Glu 28 present at the flavin active site in catalysis of folate binding and reduction [Trimmer01a]. Asp120 is not critical for flavin reduction by NADH [Trimmer05]. Asp120 adopts "open" and "closed" conformations to ensure differential recognition of NADH and folate [Pejchal05]. Conformationally mobile Phe223 or Leu223 plays role in both NADH and folate binding [Lee09c].

Cofactors or Prosthetic Groups: FAD [Comment 5, Sheppard99]

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
NADH
13.0
[Sheppard99]
NADH
17.0
23.5
[Guenther99, BRENDA14]
NADH
20.0, 66.0
10.4, 55.0
[Lee09c, BRENDA14]
a 5,10-methylene-tetrahydrofolate
0.5
[Lee09c, BRENDA14]
a 5,10-methylene-tetrahydrofolate
3.9
[Guenther99, BRENDA14]
a 5,10-methylene-tetrahydrofolate
0.8
[Sheppard99]
an N5-methyl-tetrahydrofolate
85.0
[Trimmer01a, BRENDA14]

pH(opt): 6.3 [BRENDA14, KATZEN65], 7.2 [Sheppard99]


Sequence Features

Feature Class Location Attached Group Citations Comment
Mutagenesis-Variant 28  
[Pejchal05, Trimmer01a, UniProt14]
Alternate sequence: E → Q; UniProt: Abolishes enzyme activity.
Active-Site 28  
[Pejchal05, Trimmer01a, UniProt14]
UniProt: Proton donor/acceptor.
Nucleotide-Phosphate-Binding-Region 28 -> 33 NAD+
[UniProt14]
UniProt: NAD.
Nucleotide-Phosphate-Binding-Region 59 -> 60 NAD+
[UniProt14]
UniProt: NAD.
Nucleotide-Phosphate-Binding-Region 59 -> 62 FAD
[UniProt14]
UniProt: FAD.
Amino-Acid-Sites-That-Bind 88  
[UniProt13]
UniProt: FAD.
Nucleotide-Phosphate-Binding-Region 118 -> 120 FAD
[UniProt14]
UniProt: FAD.
Mutagenesis-Variant 120  
[Trimmer01a, UniProt14]
Alternate sequence: D → N; UniProt: Strongly reduces enzyme activity. Strongly reduces affinity for 5- methyltetrahydrofolate.
Amino-Acid-Sites-That-Bind 120  
[UniProt14]
UniProt: Substrate.
Nucleotide-Phosphate-Binding-Region 131 -> 132 FAD
[UniProt14]
UniProt: FAD.
Amino-Acid-Sites-That-Bind 152  
[UniProt13]
UniProt: FAD.
Nucleotide-Phosphate-Binding-Region 156 -> 159 FAD
[UniProt14]
UniProt: FAD.
Nucleotide-Phosphate-Binding-Region 165 -> 172 FAD
[UniProt14]
UniProt: FAD.
Mutagenesis-Variant 177  
[Guenther99, UniProt13]
Alternate sequence: A → V; UniProt: Increases the propensity to lose its essential flavin cofactor.
Amino-Acid-Sites-That-Bind 183  
[UniProt14]
UniProt: Substrate.
Amino-Acid-Sites-That-Bind 219  
[UniProt14]
UniProt: Substrate.
Mutagenesis-Variant 223  
[Lee09c, UniProt14]
Alternate sequence: F → L; UniProt: Slightly reduced enzyme activity.
Alternate sequence: F → A; UniProt: Strongly decreases substrate and NADH binding.
Amino-Acid-Sites-That-Bind 223  
[UniProt14]
UniProt: Substrate.
Amino-Acid-Sites-That-Bind 275  
[UniProt14]
UniProt: Substrate.
Amino-Acid-Sites-That-Bind 279  
[UniProt14]
UniProt: Substrate.


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b3941 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10585; confirmed by SwissProt match.


References

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Belfaiza87: Belfaiza J, Guillou Y, Margarita D, Perrin D, Saint Girons I (1987). "Operator-constitutive mutations of the Escherichia coli metF gene." J Bacteriol 1987;169(2);670-4. PMID: 3542965

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

Cai92: Cai XY, Jakubowski H, Redfield B, Zaleski B, Brot N, Weissbach H (1992). "Role of the metF and metJ genes on the vitamin B12 regulation of methionine gene expression: involvement of N5-methyltetrahydrofolic acid." Biochem Biophys Res Commun 1992;182(2);651-8. PMID: 1734876

Cathou63: Cathou RE, Buchanan JM "Enzymatic Synthesis of the Methyl Group of Methionine V. Studies with 5,10-methylenetetrahydrofolate reductase from Escherichia coli." The Journal of Biological Chemistry 238: 1746-1751 (1963).

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Donaldson62: Donaldson KO, Keresztesy JC "Naturally Occurring Forms of Folic Acid II. Enzymatic conversion of methylenetetrahydrofolic acid to prefolic A-methyltetrahydrofolate." The Journal of Biological Chemistry 237: 1298-1304 (1962).

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

Greene84: Greene RC, Smith AA (1984). "Insertion mutagenesis of the metJBLF gene cluster of Escherichia coli K-12: evidence for an metBL operon." J Bacteriol 1984;159(2);767-9. PMID: 6086586

Guenther99: Guenther BD, Sheppard CA, Tran P, Rozen R, Matthews RG, Ludwig ML (1999). "The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia." Nat Struct Biol 6(4);359-65. PMID: 10201405

Guest64a: Guest JR, Foster MA, Woods DD "Methyl Derivatives of Folic Acid as Intermediates in the Methylation of Homocysteine by Escherichia coli." Biochemical Journal 92: 488-496 (1964).

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

KATZEN65: KATZEN HM, BUCHANAN JM (1965). "ENZYMATIC SYNTHESIS OF THE METHYL GROUP OF METHIONINE. 8. REPRESSION-DEREPRESSION, PURIFICATION, AND PROPERTIES OF 5,10-METHYLENETETRAHYDROFOLATE REDUCTASE FROM ESCHERICHIA COLI." J Biol Chem 240;825-35. PMID: 14275142

Katzen65: Katzen HM, Buchanan JM "Enzymatic Synthesis of the Methyl Group of Methionine VIII. Repression-depression, purification and properties of 5,10-methylene- tetrahydrofolate reductase from Escherichia coli." The Journal of Biological Chemistry 240: 825-835 (1965).

Kerner05: Kerner MJ, Naylor DJ, Ishihama Y, Maier T, Chang HC, Stines AP, Georgopoulos C, Frishman D, Hayer-Hartl M, Mann M, Hartl FU (2005). "Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli." Cell 122(2);209-20. PMID: 16051146

Larrabee63: Larrabee AR, Rosenthal S, Cathou RE, Buchanan JM "Enzymatic Synthesis of the Methyl Group of Methionine IV. Isolation, characterization and role of 5-methyl tetrahydrofolate." The Journal of Biological Chemistry 238: 1025-1031 (1963).

Lee09c: Lee MN, Takawira D, Nikolova AP, Ballou DP, Furtado VC, Phung NL, Still BR, Thorstad MK, Tanner JJ, Trimmer EE (2009). "Functional role for the conformationally mobile phenylalanine 223 in the reaction of methylenetetrahydrofolate reductase from Escherichia coli." Biochemistry 48(32);7673-85. PMID: 19610625

Misra03: Misra SK, Bhakuni V (2003). "Unique holoenzyme dimers of the tetrameric enzyme Escherichia coli methylenetetrahydrofolate reductase: characterization of structural features associated with modulation of the enzyme's function." Biochemistry 42(13);3921-8. PMID: 12667083

Neidhardt96: Neidhardt FC, Curtiss III R, Ingraham JL, Lin ECC, Low Jr KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE "Escherichia coli and Salmonella, Cellular and Molecular Biology, Second Edition." American Society for Microbiology, Washington, D.C., 1996.

Patrick07: Patrick WM, Quandt EM, Swartzlander DB, Matsumura I (2007). "Multicopy suppression underpins metabolic evolvability." Mol Biol Evol 24(12);2716-22. PMID: 17884825

Pejchal05: Pejchal R, Sargeant R, Ludwig ML (2005). "Structures of NADH and CH3-H4folate complexes of Escherichia coli methylenetetrahydrofolate reductase reveal a spartan strategy for a ping-pong reaction." Biochemistry 44(34);11447-57. PMID: 16114881

SaintGirons83: Saint-Girons I, Duchange N, Zakin MM, Park I, Margarita D, Ferrara P, Cohen GN (1983). "Nucleotide sequence of metF, the E. coli structural gene for 5-10 methylene tetrahydrofolate reductase and of its control region." Nucleic Acids Res 1983;11(19);6723-32. PMID: 6356036

Sheppard99: Sheppard CA, Trimmer EE, Matthews RG (1999). "Purification and properties of NADH-dependent 5, 10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli." J Bacteriol 1999;181(3);718-25. PMID: 9922232

Trimmer01: Trimmer EE, Ballou DP, Matthews RG (2001). "Methylenetetrahydrofolate reductase from Escherichia coli: elucidation of the kinetic mechanism by steady-state and rapid-reaction studies." Biochemistry 40(21);6205-15. PMID: 11371181

Trimmer01a: Trimmer EE, Ballou DP, Ludwig ML, Matthews RG (2001). "Folate activation and catalysis in methylenetetrahydrofolate reductase from Escherichia coli: roles for aspartate 120 and glutamate 28." Biochemistry 40(21);6216-26. PMID: 11371182

Trimmer05: Trimmer EE, Ballou DP, Galloway LJ, Scannell SA, Brinker DR, Casas KR (2005). "Aspartate 120 of Escherichia coli methylenetetrahydrofolate reductase: evidence for major roles in folate binding and catalysis and a minor role in flavin reactivity." Biochemistry 44(18);6809-22. PMID: 15865426

UniProt13: UniProt Consortium (2013). "UniProt version 2013-08 released on 2013-08-01 00:00:00." Database.

UniProt14: UniProt Consortium (2014). "UniProt version 2014-01 released on 2014-01-01 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Other References Related to Gene Regulation

Liu01: Liu R, Blackwell TW, States DJ (2001). "Conformational model for binding site recognition by the E.coli MetJ transcription factor." Bioinformatics 17(7);622-33. PMID: 11448880

Stauffer88: Stauffer GV, Stauffer LT (1988). "Salmonella typhimurium LT2 metF operator mutations." Mol Gen Genet 1988;214(1);32-6. PMID: 3147373


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 18.5 on Sun Nov 23, 2014, BIOCYC13B.