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Escherichia coli K-12 substr. MG1655 Enzyme: ornithine carbamoyltransferase chain F



Gene: argF Accession Numbers: EG10067 (EcoCyc), b0273, ECK0274

Synonyms: OTCase-2

Regulation Summary Diagram: ?

Subunit composition of ornithine carbamoyltransferase chain F = [ArgF]3

Summary:
E. coli K-12 contains two structural genes, argF and argI, encoding ornithine carbamoyltransferase, both of which catalyze the sixth step of arginine biosynthesis. The active enzyme molecule is a trimer, and the products of the two genes become associated in a family of four isoenzymes, namely FFF, FFI, FII, and III. As compared to argI, argF might be the product of a relatively recent event of duplication or transposition in E. coli K-12 [Legrain76a].

Ornithine carbamoyltransferase catalyzes the synthesis of L-citrulline by transfer of the carbamoyl group from carbamoyl phosphate to L-ornithine. Zn2+ is a homotropic effector and an allosteric cofactor of ornithine transcarbamoylase [Kuo82].

Arg57 of the enzyme is essential for the tight binding to carbamoyl phosphate and the productive binding to L-ornithine for efficient catalysis [Kuo88]. A carbamoyl phosphate-induced isomerization of ornithine transcarbamoylase causes substrate binding to be ordered. It leads to a random substrate-binding pattern if Arg57 in the active site of the enzyme is replaced by a glycine [Kuo89]. The enzyme follows an ordered Bi Bi mechanism with carbamoyl phosphate being the first substrate bound and L-citrulline being the first product released [Legrain76]. Substrate binding order of this enzyme is dictated by the protonation state of Arg57. The positive charge at Arg57 enhances the rate of carbamoyl phosphate binding, augments the turnover rate and controls the substrate binding order of carbamoylation. Carbamoyl phosphate-induced conformational change is the rate-limiting step of the enzymic reaction in the forward direction [Goldsmith93]. The hydrogen-bonding interactions of carbamoyl phosphate to the enzyme reduce the rate of thermal decomposition of carbamoyl phosphate by a factor of >5,000 by preventing the C-O bond breakage [Wang08b].

The crystal structure of the enzyme has been determined at 2.8 Å resolution [Jin97] and of the enzyme in complex with substrate analogs at 2.8 Å resolution [Ha97] and 1.8 Å resolution [Langley00].

Gene Citations: [Cunin83]

Locations: cytosol

Map Position: [288,525 <- 289,529] (6.22 centisomes)
Length: 1005 bp / 334 aa

Molecular Weight of Polypeptide: 36.827 kD (from nucleotide sequence)

Isozyme Sequence Similarity [Comment 1]:
ArgI: YES

Unification Links: ASAP:ABE-0000939 , CGSC:1016 , EchoBASE:EB0065 , EcoGene:EG10067 , EcoliWiki:b0273 , Entrez-gene:944844 , ModBase:P06960 , OU-Microarray:b0273 , PortEco:argF , PR:PRO_000022130 , Protein Model Portal:P06960 , RefSeq:NP_414807 , RegulonDB:EG10067 , SMR:P06960 , String:511145.b0273 , Swiss-Model:P06960 , UniProt:P06960

Relationship Links: InterPro:IN-FAMILY:IPR002292 , InterPro:IN-FAMILY:IPR006130 , InterPro:IN-FAMILY:IPR006131 , InterPro:IN-FAMILY:IPR006132 , InterPro:IN-FAMILY:IPR024904 , Pfam:IN-FAMILY:PF00185 , Pfam:IN-FAMILY:PF02729 , Prints:IN-FAMILY:PR00100 , Prints:IN-FAMILY:PR00102 , Prosite:IN-FAMILY:PS00097

In Paralogous Gene Group: 84 (4 members)

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0006974 - cellular response to DNA damage stimulus Inferred from experiment [Khil02]
GO:0006520 - cellular amino acid metabolic process Inferred by computational analysis [GOA01]
GO:0006526 - arginine biosynthetic process Inferred by computational analysis [UniProtGOA12, UniProtGOA11, GOA06]
GO:0006591 - ornithine metabolic process Inferred by computational analysis [GOA01]
GO:0008652 - cellular amino acid biosynthetic process Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0004585 - ornithine carbamoyltransferase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Legrain72]
GO:0016597 - amino acid binding Inferred by computational analysis [GOA01]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
GO:0016743 - carboxyl- or carbamoyltransferase activity Inferred by computational analysis [GOA01]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11a, UniProtGOA11, GOA06]
GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: extrachromosomal prophage genes and phage related functions
metabolism biosynthesis of building blocks amino acids arginine

Essentiality data for argF knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 2]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 3]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 4]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 3]
Yes [Feist07, Comment 5]

Credits:
Last-Curated ? 30-Mar-2011 by Fulcher C , SRI International


Enzymatic reaction of: ornithine carbamoyltransferase

Synonyms: ornithine transcarbamylase, citrulline phosphorylase, OTC

EC Number: 2.1.3.3

L-ornithine + carbamoyl-phosphate <=> L-citrulline + phosphate + 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. [Legrain76a]

In Pathways: superpathway of arginine and polyamine biosynthesis , arginine biosynthesis I (via L-ornithine)

Summary:
Isomerization of the enzyme occurs upon the binding of the substrate carbamoyl phosphate. This is essential for the stereospecific addition of the second substrate L-ornithine. Arg-57 mutant shows no isomerization. [Kuo89] Substrates bind in order, phosphate is the last product to be released. [Legrain76] Arg-57 is critical for isomerization and ornithine binding [Kuo89, Kuo88]

Inhibitors (Competitive): Zn2+ [Comment 6]

Inhibitors (Unknown Mechanism): a sulfhydryl reagent [Vogel74] , N-δ-(phosphonoacetyl)-L-ornithine [Penninckx79]

Kinetic Parameters:

Substrate
Km (μM)
Citations
carbamoyl-phosphate
60.0, 360.0
[Baur90, BRENDA14]


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Falmagne85, UniProt11]
UniProt: Removed.
Chain 2 -> 334
[UniProt09]
UniProt: Ornithine carbamoyltransferase chain F;
Amino-Acid-Sites-That-Bind 10
[UniProt12b]
UniProt: Carbamoyl phosphate; Non-Experimental Qualifier: by similarity.
Amino-Acid-Site 31
[UniProt10]
UniProt: Important for structural integrity; Sequence Annotation Type: site; Non-Experimental Qualifier: by similarity;
Protein-Segment 56 -> 60
[UniProt10]
UniProt: Carbamoyl phosphate binding; Sequence Annotation Type: region of interest; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 72
[UniProt12b]
UniProt: Carbamoyl phosphate; Non-Experimental Qualifier: by similarity.
Amino-Acid-Sites-That-Bind 83
[UniProt12b]
UniProt: Carbamoyl phosphate; Non-Experimental Qualifier: by similarity.
Amino-Acid-Sites-That-Bind 107
[UniProt10]
UniProt: Carbamoyl phosphate; Non-Experimental Qualifier: by similarity;
Sequence-Conflict 121
[Van84, UniProt10a]
Alternate sequence: A → P; UniProt: (in Ref. 1; CAA25329);
Protein-Segment 134 -> 137
[UniProt12b]
UniProt: Carbamoyl phosphate binding; Sequence Annotation Type: region of interest; Non-Experimental Qualifier: by similarity.
Amino-Acid-Site 147
[UniProt10]
UniProt: Important for structural integrity; Sequence Annotation Type: site; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 168
[UniProt12b]
UniProt: Ornithine; Non-Experimental Qualifier: by similarity.
Amino-Acid-Sites-That-Bind 232
[UniProt12b]
UniProt: Ornithine; Non-Experimental Qualifier: by similarity.
Protein-Segment 236 -> 237
[UniProt12b]
UniProt: Ornithine binding; Sequence Annotation Type: region of interest; Non-Experimental Qualifier: by similarity.
Protein-Segment 273 -> 276
[UniProt10]
UniProt: Ornithine binding; Sequence Annotation Type: region of interest; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 274
[UniProt12b]
UniProt: Zinc; Non-Experimental Qualifier: by similarity.
Amino-Acid-Sites-That-Bind 302
[UniProt12b]
UniProt: Carbamoyl phosphate; Non-Experimental Qualifier: by similarity.
Amino-Acid-Sites-That-Bind 320
[UniProt12b]
UniProt: Carbamoyl phosphate; Non-Experimental Qualifier: by similarity.


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b0273 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10067; 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

Baur90: Baur H, Tricot C, Stalon V, Haas D (1990). "Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme." J Biol Chem 265(25);14728-31. PMID: 2118516

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

Cunin83: Cunin R, Eckhardt T, Piette J, Boyen A, Pierard A, Glansdorff N (1983). "Molecular basis for modulated regulation of gene expression in the arginine regulon of Escherichia coli K-12." Nucleic Acids Res 1983;11(15);5007-19. PMID: 6348703

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

Falmagne85: Falmagne P, Portetelle D, Stalon V (1985). "Immunological and structural relatedness of catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria." J Bacteriol 161(2);714-9. PMID: 3968036

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.

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Goldsmith93: Goldsmith JO, Kuo LC (1993). "Protonation of arginine 57 of Escherichia coli ornithine transcarbamoylase regulates substrate binding and turnover." J Biol Chem 268(25);18485-90. PMID: 8395503

Ha97: Ha Y, McCann MT, Tuchman M, Allewell NM (1997). "Substrate-induced conformational change in a trimeric ornithine transcarbamoylase." Proc Natl Acad Sci U S A 94(18);9550-5. PMID: 9275160

Jin97: Jin L, Seaton BA, Head JF (1997). "Crystal structure at 2.8 A resolution of anabolic ornithine transcarbamylase from Escherichia coli." Nat Struct Biol 4(8);622-5. PMID: 9253409

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

Kuo82: Kuo LC, Lipscomb WN, Kantrowitz ER (1982). "Zn(II)-induced cooperativity of Escherichia coli ornithine transcarbamoylase." Proc Natl Acad Sci U S A 1982;79(7);2250-4. PMID: 7048313

Kuo83: Kuo LC "Allosteric cofactor-mediated enzyme cooperativity: A theoretical treatment." Proc Natl Acad Sci USA 1983;80:3243-3247.

Kuo88: Kuo LC, Miller AW, Lee S, Kozuma C (1988). "Site-directed mutagenesis of Escherichia coli ornithine transcarbamoylase: role of arginine-57 in substrate binding and catalysis." Biochemistry 1988;27(24);8823-32. PMID: 3072022

Kuo89: Kuo LC, Seaton BA (1989). "X-ray diffraction analysis on single crystals of recombinant Escherichia coli ornithine transcarbamoylase." J Biol Chem 1989;264(27);16246-8. PMID: 2674127

Langley00: Langley DB, Templeton MD, Fields BA, Mitchell RE, Collyer CA (2000). "Mechanism of inactivation of ornithine transcarbamoylase by Ndelta -(N'-Sulfodiaminophosphinyl)-L-ornithine, a true transition state analogue? Crystal structure and implications for catalytic mechanism." J Biol Chem 275(26);20012-9. PMID: 10747936

Legrain72: Legrain C, Halleux P, Stalon V, Glansdorff N (1972). "The dual genetic control of ornithine carbamolytransferase in Escherichia coli. A case of bacterial hybrid enzymes." Eur J Biochem 1972;27(1);93-102. PMID: 4558857

Legrain76: Legrain C, Stalon V (1976). "Ornithine carbamoyltransferase from Escherichia coli W. Purification, structure and steady-state kinetic analysis." Eur J Biochem 1976;63(1);289-301. PMID: 4319

Legrain76a: Legrain C, Stalon V, Glansdorff N (1976). "Escherichia coli ornithine carbamolytransferase isoenzymes: evolutionary significance and the isolation of lambdaargF and lambdaargI transducing bacteriophages." J Bacteriol 1976;128(1);35-8. PMID: 789338

Penninckx79: Penninckx M, Gigot D (1979). "Synthesis of a peptide form of N-delta-(phosphonoacetyl)-L-ornithine. Its antibacterial effect through the specific inhibition of Escherichia coli L-ornithine carbamoyltransferase." J Biol Chem 1979;254(14);6392-6. PMID: 376531

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

UniProt10: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProt12b: UniProt Consortium (2012). "UniProt version 2012-11 released on 2012-11-26 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."

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

Van: Van Vliet F, Boyen A, Glansdorff N "On interspecies gene transfer: the case of the argF gene of Escherichia coli." Ann Inst Pasteur Microbiol NIL;139(4);493-6. PMID: 3052532

Van84: Van Vliet F, Cunin R, Jacobs A, Piette J, Gigot D, Lauwereys M, Pierard A, Glansdorff N (1984). "Evolutionary divergence of genes for ornithine and aspartate carbamoyl-transferases--complete sequence and mode of regulation of the Escherichia coli argF gene; comparison of argF with argI and pyrB." Nucleic Acids Res 1984;12(15);6277-89. PMID: 6382166

Vogel74: Vogel HJ, Vogel RH (1974). "Enzymes of arginine biosynthesis and their repressive control." Adv Enzymol Relat Areas Mol Biol 1974;40(0);65-90. PMID: 4365537

Wang08b: Wang Q, Xia J, Guallar V, Krilov G, Kantrowitz ER (2008). "Mechanism of thermal decomposition of carbamoyl phosphate and its stabilization by aspartate and ornithine transcarbamoylases." Proc Natl Acad Sci U S A 105(44);16918-23. PMID: 18971327

Other References Related to Gene Regulation

Caldara06: Caldara M, Charlier D, Cunin R (2006). "The arginine regulon of Escherichia coli: whole-system transcriptome analysis discovers new genes and provides an integrated view of arginine regulation." Microbiology 152(Pt 11);3343-54. PMID: 17074904

Charlier92: Charlier D, Roovers M, Van Vliet F, Boyen A, Cunin R, Nakamura Y, Glansdorff N, Pierard A (1992). "Arginine regulon of Escherichia coli K-12. A study of repressor-operator interactions and of in vitro binding affinities versus in vivo repression." J Mol Biol 1992;226(2);367-86. PMID: 1640456

Levitt92: Levitt M (1992). "Accurate modeling of protein conformation by automatic segment matching." J Mol Biol 226(2);507-33. PMID: 1640463

Makarova01: Makarova KS, Mironov AA, Gelfand MS (2001). "Conservation of the binding site for the arginine repressor in all bacterial lineages." Genome Biol 2(4);RESEARCH0013. PMID: 11305941

Moore78: Moore SK, James E, James PM, Fareed G (1978). "Isolation, cloning and characterization of argF gene DNA from Escherichia coli K-12." Gene 4(3);261-78. PMID: 369953

Piette82: Piette J, Cunin R, Van Vliet F, Charlier D, Crabeel M, Ota Y, Glansdorff N (1982). "Homologous control sites and DNA transcription starts in the related argF and argI genes of Escherichia coli K12." EMBO J 1(7);853-7. PMID: 6329710

Tian92: Tian G, Lim D, Carey J, Maas WK (1992). "Binding of the arginine repressor of Escherichia coli K12 to its operator sites." J Mol Biol 226(2);387-97. PMID: 1640457

Tian94: Tian G, Maas WK (1994). "Mutational analysis of the arginine repressor of Escherichia coli." Mol Microbiol 1994;13(4);599-608. PMID: 7997172


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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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