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Escherichia coli K-12 substr. MG1655 Enzyme: D-alanyl-D-alanine-adding enzyme



Gene: murF Accession Numbers: EG10622 (EcoCyc), b0086, ECK0087

Synonyms: mra

Regulation Summary Diagram: ?

Summary:
UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopimelate-D-alanyl-D-alanine ligase (MurF) catalyzes the final cytoplasmic step in the biosynthesis of the peptidoglycan precursor [Duncan90].

Data from kinetic characterization of the enzyme is consistent with a sequential ordered reaction mechanism, with sequential binding of ATP, tripeptide, and D-Ala-D-Ala [Anderson96]. Carbamylation of the Lys202 residue is important for catalytic activity [Dementin01].

A crystal structure of the apo-enzyme has been solved at 2.3Å resolution [Yan99, Yan00].

A temperature-sensitive murF mutant has been isolated [Lugtenberg72]. The murF2 allele was determined to carry an A288T mutation in a conserved region of the enzyme; the mutant is 181-fold less catalytically active at the permissive temperature, with even lower activity at the nonpermissive temperature. Additional site-directed mutants in the conserved E158 and H188 residues have strongly reduced catalytic activity [Eveland97].

MurF is a potential antimicrobial drug target; inhibitors of MurF activity have been identified [Baum06, Baum07, Baum09, Sova09, Hrast13].

Reviews: [El03, Typas12]

Citations: [Maruyama88, Schouten06, Murakami09, Miyakawa72]

Gene Citations: [Vicente98, MenginLecreulx98, Hara97]

Locations: cytosol

Map Position: [94,650 -> 96,008] (2.04 centisomes)
Length: 1359 bp / 452 aa

Molecular Weight of Polypeptide: 47.447 kD (from nucleotide sequence), 49.0 kD (experimental) [Duncan90 ]

pI: 5.61

Unification Links: ASAP:ABE-0000313 , CGSC:474 , DIP:DIP-10281N , EchoBASE:EB0617 , EcoGene:EG10622 , EcoliWiki:b0086 , Mint:MINT-1227223 , ModBase:P11880 , OU-Microarray:b0086 , PortEco:murF , PR:PRO_000023317 , Pride:P11880 , Protein Model Portal:P11880 , RefSeq:NP_414628 , RegulonDB:EG10622 , SMR:P11880 , String:511145.b0086 , UniProt:P11880

Relationship Links: InterPro:IN-FAMILY:IPR000713 , InterPro:IN-FAMILY:IPR004101 , InterPro:IN-FAMILY:IPR005863 , InterPro:IN-FAMILY:IPR013221 , Panther:IN-FAMILY:PTHR23135:SF3 , PDB:Structure:1GG4 , Pfam:IN-FAMILY:PF01225 , Pfam:IN-FAMILY:PF02875 , Pfam:IN-FAMILY:PF08245

In Paralogous Gene Group: 32 (3 members)

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0009252 - peptidoglycan biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, UniProtGOA11a, Lugtenberg72]
GO:0007049 - cell cycle Inferred by computational analysis [UniProtGOA11a]
GO:0008360 - regulation of cell shape Inferred by computational analysis [UniProtGOA11a, GOA01]
GO:0009058 - biosynthetic process Inferred by computational analysis [GOA01]
GO:0051301 - cell division Inferred by computational analysis [UniProtGOA11a, GOA01]
GO:0071555 - cell wall organization Inferred by computational analysis [UniProtGOA11a]
Molecular Function: GO:0008766 - UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopimelate-D-alanyl-D-alanine ligase activity Inferred from experiment Inferred by computational analysis [GOA01, Duncan90]
GO:0047480 - UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase activity Inferred from experiment Inferred by computational analysis [GOA01a, Baum06, Duncan90]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11a, GOA01]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11a, GOA01]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA01]

MultiFun Terms: cell processes cell division
cell structure murein
metabolism biosynthesis of macromolecules (cellular constituents) murein (peptidoglycan)
metabolism central intermediary metabolism murein turnover, recycling

Essentiality data for murF 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 No 37 Aerobic 7   No [Baba06, Comment 2]

Credits:
Last-Curated ? 25-Jul-2013 by Keseler I , SRI International


Enzymatic reaction of: D-alanyl-D-alanine-adding enzyme

Synonyms: UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopimelate-D-alanyl-D-alanine ligase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimeloyl-D-alanyl-D-alanine synthetase, UDP-MurNAc-pentapeptide synthetase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminoheptanedioate:D-alanyl-D-alanine ligase (ADP-forming), UDP-MurNac-pentapeptide presynthetase

EC Number: 6.3.2.10

D-alanyl-D-alanine + UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-2,6-diaminopimelate + ATP <=> UDP-N-acetyl-α-D-muramoyl-L-alanyl-γ-D-glutamyl-meso-2,6-diaminopimeloyl-D-alanyl-D-alanine + ADP + phosphate + H+

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

The reaction is physiologically favored in the direction shown.

Alternative Substrates for UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-2,6-diaminopimelate: UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-L-lysine [Baum06 ] , DDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminoheptanedioate [Michaud87 ]

In Pathways: peptidoglycan biosynthesis I (meso-diaminopimelate containing) , UDP-N-acetylmuramoyl-pentapeptide biosynthesis I (meso-DAP-containing)

Summary:
The enzyme was first purified from E. coli K235 [Comb62].

Several potential dipeptide substrates have been tested [Duncan90]. In addition, the enzyme can efficiently utilize UDP-MurNAc-tripeptide substrates that contain meso-lanthionine or L-allo-cystathionine in place of meso-DAP [MenginLecreulx94a].

Inhibitors (Unknown Mechanism): a DDP-N-acetylmuramoyl-pentapeptide , a UDP-N-acetylmuramoyl-pentapeptide

Kinetic Parameters:

Substrate
Km (μM)
Citations
D-alanyl-D-alanine
100.0
[Michaud87]
ATP
164.0
[Anderson96]
UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-2,6-diaminopimelate
11.0
[Michaud87]
DDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminoheptanedioate
25.0
[Michaud87]


Sequence Features

Feature Class Location Citations Comment
Sequence-Conflict 61
[Parquet89, Yura92, UniProt10]
Alternate sequence: G → A; UniProt: (in Ref. 1 and 3);
Nucleotide-Phosphate-Binding-Region 107 -> 113
[UniProt10a]
UniProt: ATP; Non-Experimental Qualifier: potential;
Sequence-Conflict 178
[Parquet89, Yura92, UniProt10]
Alternate sequence: A → R; UniProt: (in Ref. 1 and 3);
Extrinsic-Sequence-Variant 288
[UniProt10]
Alternate sequence: A → T; UniProt: (in murF2; temperature-sensitive mutant with low activity);


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
10/20/97 Gene b0086 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10622; confirmed by SwissProt match.


References

Anderson96: Anderson MS, Eveland SS, Onishi HR, Pompliano DL (1996). "Kinetic mechanism of the Escherichia coli UDPMurNAc-tripeptide D-alanyl-D-alanine-adding enzyme: use of a glutathione S-transferase fusion." Biochemistry 35(50);16264-9. PMID: 8973200

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

Baum06: Baum EZ, Crespo-Carbone SM, Abbanat D, Foleno B, Maden A, Goldschmidt R, Bush K (2006). "Utility of muropeptide ligase for identification of inhibitors of the cell wall biosynthesis enzyme MurF." Antimicrob Agents Chemother 50(1);230-6. PMID: 16377691

Baum07: Baum EZ, Crespo-Carbone SM, Klinger A, Foleno BD, Turchi I, Macielag M, Bush K (2007). "A MurF inhibitor that disrupts cell wall biosynthesis in Escherichia coli." Antimicrob Agents Chemother 51(12);4420-6. PMID: 17908943

Baum09: Baum EZ, Crespo-Carbone SM, Foleno BD, Simon LD, Guillemont J, Macielag M, Bush K (2009). "MurF inhibitors with antibacterial activity: effect on muropeptide levels." Antimicrob Agents Chemother 53(8);3240-7. PMID: 19470511

Comb62: Comb DG (1962). "The enzymatic addition of D-alanyl-D-alanine to a uridine nucleotide-peptide." J Biol Chem 237;1601-4. PMID: 13880737

Dementin01: Dementin S, Bouhss A, Auger G, Parquet C, Mengin-Lecreulx D, Dideberg O, van Heijenoort J, Blanot D (2001). "Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments." Eur J Biochem 268(22);5800-7. PMID: 11722566

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

Duncan90: Duncan K, van Heijenoort J, Walsh CT (1990). "Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli." Biochemistry 1990;29(9);2379-86. PMID: 2186811

El03: El Zoeiby A, Sanschagrin F, Levesque RC (2003). "Structure and function of the Mur enzymes: development of novel inhibitors." Mol Microbiol 47(1);1-12. PMID: 12492849

Eveland97: Eveland SS, Pompliano DL, Anderson MS (1997). "Conditionally lethal Escherichia coli murein mutants contain point defects that map to regions conserved among murein and folyl poly-gamma-glutamate ligases: identification of a ligase superfamily." Biochemistry 36(20);6223-9. PMID: 9166795

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.

Hara97: Hara H, Yasuda S, Horiuchi K, Park JT (1997). "A promoter for the first nine genes of the Escherichia coli mra cluster of cell division and cell envelope biosynthesis genes, including ftsI and ftsW." J Bacteriol 179(18);5802-11. PMID: 9294438

Hrast13: Hrast M, Turk S, Sosič I, Knez D, Randall CP, Barreteau H, Contreras-Martel C, Dessen A, O'Neill AJ, Mengin-Lecreulx D, Blanot D, Gobec S (2013). "Structure-activity relationships of new cyanothiophene inhibitors of the essential peptidoglycan biosynthesis enzyme MurF." Eur J Med Chem 66C;32-45. PMID: 23786712

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Lugtenberg72: Lugtenberg EJ, v Schijndel-van Dam A (1972). "Temperature-sensitive mutants of Escherichia coli K-12 with low activities of the L-alanine adding enzyme and the D-alanyl-D-alanine adding enzyme." J Bacteriol 110(1);35-40. PMID: 4552998

Maruyama88: Maruyama IN, Yamamoto AH, Hirota Y (1988). "Determination of gene products and coding regions from the murE-murF region of Escherichia coli." J Bacteriol 170(8);3786-8. PMID: 2841306

MenginLecreulx94a: Mengin-Lecreulx D, Blanot D, van Heijenoort J (1994). "Replacement of diaminopimelic acid by cystathionine or lanthionine in the peptidoglycan of Escherichia coli." J Bacteriol 1994;176(14);4321-7. PMID: 8021219

MenginLecreulx98: Mengin-Lecreulx D, Ayala J, Bouhss A, van Heijenoort J, Parquet C, Hara H (1998). "Contribution of the Pmra promoter to expression of genes in the Escherichia coli mra cluster of cell envelope biosynthesis and cell division genes." J Bacteriol 180(17);4406-12. PMID: 9721276

Michaud87: Michaud C, Blanot D, Flouret B, Van Heijenoort J (1987). "Partial purification and specificity studies of the D-glutamate-adding and D-alanyl-D-alanine-adding enzymes from Escherichia coli K12." Eur J Biochem 1987;166(3);631-7. PMID: 3301347

Miyakawa72: Miyakawa T, Matsuzawa H, Matsuhashi M, Sugino Y (1972). "Cell wall peptidoglycan mutants of Escherichia coli K-12: existence of two clusters of genes, mra and mrb, for cell wall peptidoglycan biosynthesis." J Bacteriol 112(2);950-8. PMID: 4563986

Murakami09: Murakami R, Muramatsu Y, Minami E, Masuda K, Sakaida Y, Endo S, Suzuki T, Ishida O, Takatsu T, Miyakoshi S, Inukai M, Isono F (2009). "A novel assay of bacterial peptidoglycan synthesis for natural product screening." J Antibiot (Tokyo) 62(3);153-8. PMID: 19229285

Parquet89: Parquet C, Flouret B, Mengin-Lecreulx D, van Heijenoort J (1989). "Nucleotide sequence of the murF gene encoding the UDP-MurNAc-pentapeptide synthetase of Escherichia coli." Nucleic Acids Res 17(13);5379. PMID: 2668880

Schouten06: Schouten JA, Bagga S, Lloyd AJ, de Pascale G, Dowson CG, Roper DI, Bugg TD (2006). "Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5." Mol Biosyst 2(10);484-91. PMID: 17216029

Sova09: Sova M, Kovac A, Turk S, Hrast M, Blanot D, Gobec S (2009). "Phosphorylated hydroxyethylamines as novel inhibitors of the bacterial cell wall biosynthesis enzymes MurC to MurF." Bioorg Chem 37(6);217-22. PMID: 19804894

Typas12: Typas A, Banzhaf M, Gross CA, Vollmer W (2012). "From the regulation of peptidoglycan synthesis to bacterial growth and morphology." Nat Rev Microbiol 10(2);123-36. PMID: 22203377

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 the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: 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."

Vicente98: Vicente M, Gomez MJ, Ayala JA (1998). "Regulation of transcription of cell division genes in the Escherichia coli dcw cluster." Cell Mol Life Sci 54(4);317-24. PMID: 9614967

Yan00: Yan Y, Munshi S, Leiting B, Anderson MS, Chrzas J, Chen Z (2000). "Crystal structure of Escherichia coli UDPMurNAc-tripeptide d-alanyl-d-alanine-adding enzyme (MurF) at 2.3 A resolution." J Mol Biol 304(3);435-45. PMID: 11090285

Yan99: Yan Y, Munshi S, Li Y, Pryor KA, Marsilio F, Leiting B (1999). "Crystallization and preliminary X-ray analysis of the Escherichia coli UDP-MurNAc-tripeptide D-alanyl-D-alanine-adding enzyme (MurF)." Acta Crystallogr D Biol Crystallogr 55(Pt 12);2033-4. PMID: 10666581

Yura92: Yura T, Mori H, Nagai H, Nagata T, Ishihama A, Fujita N, Isono K, Mizobuchi K, Nakata A (1992). "Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region." Nucleic Acids Res 1992;20(13);3305-8. PMID: 1630901

Other References Related to Gene Regulation

Eraso14: Eraso JM, Markillie LM, Mitchell HD, Taylor RC, Orr G, Margolin W (2014). "The Highly Conserved MraZ Protein Is a Transcriptional Regulator in Escherichia coli." J Bacteriol 196(11);2053-66. PMID: 24659771

Gohler11: Gohler AK, Kokpinar O, Schmidt-Heck W, Geffers R, Guthke R, Rinas U, Schuster S, Jahreis K, Kaleta C (2011). "More than just a metabolic regulator - elucidation and validation of new targets of PdhR in Escherichia coli." BMC Syst Biol 5(1);197. PMID: 22168595

Ishino89: Ishino F, Jung HK, Ikeda M, Doi M, Wachi M, Matsuhashi M (1989). "New mutations fts-36, lts-33, and ftsW clustered in the mra region of the Escherichia coli chromosome induce thermosensitive cell growth and division." J Bacteriol 171(10);5523-30. PMID: 2676977


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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 18.5 on Wed Dec 17, 2014, biocyc13.