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Escherichia coli K-12 substr. MG1655 Enzyme: pyruvate formate-lyase activating enzyme



Gene: pflA Accession Numbers: EG10028 (EcoCyc), b0902, ECK0893

Synonyms: act, PFL activase, PFL-AE

Regulation Summary Diagram: ?

Regulation summary diagram for pflA

Summary:
Under anaerobic conditions, pyruvate formate-lyase activating enzyme (PflA, PFL-AE) activates PflB by generating an essential glycyl radical within the enzyme [Knappe69, Conradt84]. PFL-AE belongs to the radical SAM superfamily of proteins [Sofia01, Nicolet04]. The enzyme is also capable of activating the tdcE-encoded 2-ketobutyrate formate-lyase / pyruvate formate-lyase 4 [Hesslinger98] and stress-induced alternate pyruvate formate-lyase subunit [Wagner01].

PFL-AE uses S-adenosylmethionine (SAM) and reduced flavodoxin as co-substrates to produce the glycyl radical, generating 5'-deoxyadenosine and methionine as side products. The pro-S hydrogen from Gly734 in pyruvate formate-lyase is abstracted by the 5'-deoxyadenosyl radical in the active site of PFL-AE and incorporated into 5'-deoxyadenosine [Frey94, Wagner99]. The interaction of the enzyme with SAM and the mechanism of radical generation has been investigated [Cosper03]. A redox-interconvertible [4Fe-4S] cluster is essential for interaction with S-adenosylmethionine [Kulzer98, Broderick00]. PFL-AE undergoes cluster interconversions in vivo in response to the redox state of the cell [Yang09a]. Site-directed mutagenesis of cysteine residues showed that Cys29, 33 and 36 are required for catalytic activity [Kulzer98]. Pyruvate or oxamate are required for optimal PFL activation [Crain14].

Crystal structures of the substrate-free and 7-mer substrate peptide-bound forms have been solved. PFL-AE undergoes a conformational change upon substrate binding [Vey08]. The conformational change is rate limiting for the interaction between PFL-AE and PFL [Crain14].

PflA expression is not significantly induced by anaerobiosis [Rodel88, Sauter90]. The fermentation products of a pflA mutant have been measured with different carbon sources and under aerobic and microaerobic conditions [Zhu04a].

Act: "pyruvate formate-lyase activating enzyme"

PflA: "pyruvate formate-lyase activating enzyme"

Reviews: [Matsuoka14, Sawers98a, Knappe90]

Citations: [Knappe76, Knappe84, Zhu05a, Hasan08, Mugabi12, Marzan13]

Locations: cytosol

Map Position: [949,563 <- 950,303] (20.47 centisomes, 74°)
Length: 741 bp / 246 aa

Molecular Weight of Polypeptide: 28.204 kD (from nucleotide sequence), 29.5 kD (experimental) [Conradt84 ]

Unification Links: ASAP:ABE-0003068 , CGSC:35839 , DIP:DIP-35915N , EchoBASE:EB0027 , EcoGene:EG10028 , EcoliWiki:b0902 , OU-Microarray:b0902 , PortEco:pflA , PR:PRO_000023516 , Pride:P0A9N4 , Protein Model Portal:P0A9N4 , RefSeq:NP_415422 , RegulonDB:EG10028 , SMR:P0A9N4 , String:511145.b0902 , UniProt:P0A9N4

Relationship Links: InterPro:IN-FAMILY:IPR001989 , InterPro:IN-FAMILY:IPR006638 , InterPro:IN-FAMILY:IPR007197 , InterPro:IN-FAMILY:IPR012838 , PDB:Structure:3C8F , PDB:Structure:3CB8 , Pfam:IN-FAMILY:PF04055 , Prosite:IN-FAMILY:PS01087 , Smart:IN-FAMILY:SM00729

In Paralogous Gene Group: 205 (7 members)

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0006974 - cellular response to DNA damage stimulus Inferred from experiment [Khil02]
GO:0018307 - enzyme active site formation Inferred from experiment [Conradt84]
GO:0005975 - carbohydrate metabolic process Inferred by computational analysis [UniProtGOA11]
GO:0006006 - glucose metabolic process Inferred by computational analysis [UniProtGOA11]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11, GOA01]
Molecular Function: GO:0005515 - protein binding Inferred from experiment [Crain13, Rajagopala14]
GO:0016491 - oxidoreductase activity Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA01, Yang09a]
GO:0043365 - [formate-C-acetyltransferase]-activating enzyme activity Inferred from experiment Inferred by computational analysis [GOA01a, GOA01, Conradt84]
GO:0051539 - 4 iron, 4 sulfur cluster binding Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA01, Kulzer98]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11]
GO:0051536 - iron-sulfur cluster binding Inferred by computational analysis [UniProtGOA11, GOA01]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, LopezCampistrou05]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11a, UniProtGOA11]

MultiFun Terms: information transfer protein related posttranslational modification
metabolism energy metabolism, carbon anaerobic respiration
regulation type of regulation posttranscriptional inhibition / activation of enzymes

Essentiality data for pflA 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 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Credits:
Last-Curated ? 04-Dec-2014 by Keseler I , SRI International


Enzymatic reaction of: pyruvate formate-lyase activating enzyme

Synonyms: activase, PFL-activating enzyme

EC Number: 1.97.1.4

pyruvate formate-lyase (inactive) + a reduced flavodoxin + S-adenosyl-L-methionine <=> 5'-deoxyadenosine + L-methionine + pyruvate formate-lyase / 2-ketobutyrate formate-lyase + an oxidized flavodoxin

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

The reaction is physiologically favored in the direction shown.

Alternative Substrates for pyruvate formate-lyase (inactive): stress-induced alternate pyruvate formate-lyase subunit [Wagner01 ]

Alternative Substrates for a reduced flavodoxin: 5-deazaflavin [Conradt84 ]

Cofactors or Prosthetic Groups: a [4Fe-4S] iron-sulfur cluster [Kulzer98, Broderick97]

Activators (Unknown Mechanism): pyruvate [Crain14]

Kinetic Parameters:

Substrate
Km (μM)
Citations
S-adenosyl-L-methionine
2.8
[Wong93, BRENDA14]
pyruvate formate-lyase (inactive)
1.2
[Wong93]


Enzymatic reaction of: pyruvate formate-lyase activating enzyme

Synonyms: activase, PFL-activating enzyme

EC Number: 1.97.1.4

2-ketobutyrate formate-lyase/pyruvate formate-lyase 4, inactive + a reduced flavodoxin + S-adenosyl-L-methionine <=> 5'-deoxyadenosine + 2-ketobutyrate formate-lyase / pyruvate formate-lyase 4 + an oxidized flavodoxin

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

Reversibility of this reaction is unspecified.

Cofactors or Prosthetic Groups: a [4Fe-4S] iron-sulfur cluster [Kulzer98, Broderick97]


Sequence Features

Protein sequence of pyruvate formate-lyase activating enzyme with features indicated

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[UniProt10, Wong93]
UniProt: Removed;
Chain 2 -> 246
[UniProt09]
UniProt: Pyruvate formate-lyase 1-activating enzyme;
Metal-Binding-Site 30
[UniProt10a]
UniProt: Iron-sulfur (4Fe-4S-S-AdoMet); Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 34
[UniProt10a]
UniProt: Iron-sulfur (4Fe-4S-S-AdoMet); Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 37
[UniProt10a]
UniProt: Iron-sulfur (4Fe-4S-S-AdoMet); Non-Experimental Qualifier: by similarity;


Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram

Notes:

History:
10/20/97 Gene b0902 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10028; 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

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

Broderick00: Broderick JB, Henshaw TF, Cheek J, Wojtuszewski K, Smith SR, Trojan MR, McGhan RM, Kopf A, Kibbey M, Broderick WE (2000). "Pyruvate formate-lyase-activating enzyme: strictly anaerobic isolation yields active enzyme containing a [3Fe-4S](+) cluster." Biochem Biophys Res Commun 269(2);451-6. PMID: 10708574

Broderick97: Broderick JB, Duderstadt RE, Fernandez DC, Wojtuszewski K, Henshaw TF, Johnson MK (1997). "Pyruvate Formate-Lyase Activating Enzyme Is an Iron-Sulfur Protein." J Am Chem Soc 119;7396-7397.

Conradt84: Conradt H, Hohmann-Berger M, Hohmann HP, Blaschkowski HP, Knappe J (1984). "Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: isolation and structural properties." Arch Biochem Biophys 1984;228(1);133-42. PMID: 6364987

Cosper03: Cosper MM, Cosper NJ, Hong W, Shokes JE, Broderick WE, Broderick JB, Johnson MK, Scott RA (2003). "Structural studies of the interaction of S-adenosylmethionine with the [4Fe-4S] clusters in biotin synthase and pyruvate formate-lyase activating enzyme." Protein Sci 12(7);1573-7. PMID: 12824504

Crain13: Crain AV, Broderick JB (2013). "Flavodoxin cofactor binding induces structural changes that are required for protein-protein interactions with NADP(+) oxidoreductase and pyruvate formate-lyase activating enzyme." Biochim Biophys Acta 1834(12);2512-9. PMID: 24016774

Crain14: Crain AV, Broderick JB (2014). "Pyruvate formate-lyase and its activation by pyruvate formate-lyase activating enzyme." J Biol Chem 289(9);5723-9. PMID: 24338017

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

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

Frey94: Frey M, Rothe M, Wagner AF, Knappe J (1994). "Adenosylmethionine-dependent synthesis of the glycyl radical in pyruvate formate-lyase by abstraction of the glycine C-2 pro-S hydrogen atom. Studies of [2H]glycine-substituted enzyme and peptides homologous to the glycine 734 site." J Biol Chem 1994;269(17);12432-7. PMID: 8175649

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.

Hasan08: Hasan CM, Shimizu K (2008). "Effect of temperature up-shift on fermentation and metabolic characteristics in view of gene expressions in Escherichia coli." Microb Cell Fact 7;35. PMID: 19055729

Hesslinger98: Hesslinger C, Fairhurst SA, Sawers G (1998). "Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate." Mol Microbiol 1998;27(2);477-92. PMID: 9484901

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

Khil02: Khil PP, Camerini-Otero RD (2002). "Over 1000 genes are involved in the DNA damage response of Escherichia coli." Mol Microbiol 44(1);89-105. PMID: 11967071

Knappe69: Knappe J, Schacht J, Mockel W, Hopner T, Vetter H, Edenharder R (1969). "Pyruvate formate-lyase reaction in Escherichia coli. The enzymatic system converting an inactive form of the lyase into the catalytically active enzyme." Eur J Biochem 11(2);316-27. PMID: 4902610

Knappe76: Knappe J, Schmitt T (1976). "A novel reaction of S-adenosyl-L-methionine correlated with the activation of pyruvate formate-lyase." Biochem Biophys Res Commun 71(4);1110-7. PMID: 971302

Knappe84: Knappe J, Neugebauer FA, Blaschkowski HP, Ganzler M (1984). "Post-translational activation introduces a free radical into pyruvate formate-lyase." Proc Natl Acad Sci U S A 81(5);1332-5. PMID: 6369325

Knappe90: Knappe J, Sawers G (1990). "A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli." FEMS Microbiol Rev 1990;6(4);383-98. PMID: 2248795

Kulzer98: Kulzer R, Pils T, Kappl R, Huttermann J, Knappe J (1998). "Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme. Molecular properties of the holoenzyme form." J Biol Chem 1998;273(9);4897-903. PMID: 9478932

LopezCampistrou05: Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005). "Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth." Mol Cell Proteomics 4(8);1205-9. PMID: 15911532

Marzan13: Marzan LW, Hasan CM, Shimizu K (2013). "Effect of acidic condition on the metabolic regulation of Escherichia coli and its phoB mutant." Arch Microbiol 195(3);161-71. PMID: 23274360

Matsuoka14: Matsuoka Y, Shimizu K (2014). "Metabolic flux analysis for Escherichia coli by flux balance analysis." Methods Mol Biol 1191;237-60. PMID: 25178795

Mugabi12: Mugabi R, Sandgren D, Born M, Leith I, Horne SM, Pruβ BM (2012). "The role of activated acetate intermediates in the control of Escherichia coli biofilm amounts." Webmedcentral 3(7). PMID: 24324879

Nicolet04: Nicolet Y, Drennan CL (2004). "AdoMet radical proteins--from structure to evolution--alignment of divergent protein sequences reveals strong secondary structure element conservation." Nucleic Acids Res 32(13);4015-25. PMID: 15289575

Rajagopala14: Rajagopala SV, Sikorski P, Kumar A, Mosca R, Vlasblom J, Arnold R, Franca-Koh J, Pakala SB, Phanse S, Ceol A, Hauser R, Siszler G, Wuchty S, Emili A, Babu M, Aloy P, Pieper R, Uetz P (2014). "The binary protein-protein interaction landscape of Escherichia coli." Nat Biotechnol 32(3);285-90. PMID: 24561554

Rodel88: Rodel W, Plaga W, Frank R, Knappe J (1988). "Primary structures of Escherichia coli pyruvate formate-lyase and pyruvate-formate-lyase-activating enzyme deduced from the DNA nucleotide sequences." Eur J Biochem 177(1);153-8. PMID: 3053170

Sauter90: Sauter M, Sawers RG (1990). "Transcriptional analysis of the gene encoding pyruvate formate-lyase-activating enzyme of Escherichia coli." Mol Microbiol 4(3);355-63. PMID: 2192229

Sawers98a: Sawers G, Watson G (1998). "A glycyl radical solution: oxygen-dependent interconversion of pyruvate formate-lyase." Mol Microbiol 1998;29(4);945-54. PMID: 9767563

Sofia01: Sofia HJ, Chen G, Hetzler BG, Reyes-Spindola JF, Miller NE (2001). "Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods." Nucleic Acids Res 29(5);1097-106. PMID: 11222759

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

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

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

Vey08: Vey JL, Yang J, Li M, Broderick WE, Broderick JB, Drennan CL (2008). "Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme." Proc Natl Acad Sci U S A 105(42);16137-41. PMID: 18852451

Wagner01: Wagner AF, Schultz S, Bomke J, Pils T, Lehmann WD, Knappe J (2001). "YfiD of Escherichia coli and Y06I of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase." Biochem Biophys Res Commun 285(2);456-62. PMID: 11444864

Wagner99: Wagner AF, Demand J, Schilling G, Pils T, Knappe J (1999). "A dehydroalanyl residue can capture the 5'-deoxyadenosyl radical generated from S-adenosylmethionine by pyruvate formate-lyase-activating enzyme." Biochem Biophys Res Commun 254(2);306-10. PMID: 9918833

Wong93: Wong KK, Murray BW, Lewisch SA, Baxter MK, Ridky TW, Ulissi-DeMario L, Kozarich JW (1993). "Molecular properties of pyruvate formate-lyase activating enzyme." Biochemistry 1993;32(51);14102-10. PMID: 8260492

Yang09a: Yang J, Naik SG, Ortillo DO, Garcia-Serres R, Li M, Broderick WE, Huynh BH, Broderick JB (2009). "The iron-sulfur cluster of pyruvate formate-lyase activating enzyme in whole cells: cluster interconversion and a valence-localized [4Fe-4S]2+ state." Biochemistry 48(39);9234-41. PMID: 19711960

Zhu04a: Zhu J, Shimizu K (2004). "The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli." Appl Microbiol Biotechnol 64(3);367-75. PMID: 14673546

Zhu05a: Zhu J, Shimizu K (2005). "Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition." Metab Eng 7(2);104-15. PMID: 15781419


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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 19.0 on Sun May 24, 2015, biocyc11.