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Escherichia coli K-12 substr. MG1655 Enzyme: dihydroorotase



Gene: pyrC Accession Numbers: EG10806 (EcoCyc), b1062, ECK1047

Regulation Summary Diagram: ?

Subunit composition of dihydroorotase = [PyrC]2

Summary:
Dihydroorotase (PyrC) catalyzes the cyclization of carbamoyl-aspartate to dihydroorotate, the third reaction in the pathway for de novo biosynthesis of pyrimidine nucleotides. The enzyme is of particular interest because of its potential as an antimalarial drug target.

The structure of dihydroorotase has been determined to 1.7 Å resolution. Each subunit has a TIM motif with eight parallel β strands flanked by α helices, as well as a binuclear zinc center with two ions 3.6 Å apart [Thoden01]. A 1.9 Å structure resolved in the presence of dihydroorotate shows that a loop in PyrC is in a different conformation in each subunit of the dimer [Lee05g]. Additional crystal structures of mutant PyrC or complexed with inhibitors have been reported [Lee07h, Lee07i, Lee07d]. The surface loop structure enables productive substrate binding and stabilizes the transition state intermediate [Lee07d].

A mechanism has been proposed for the dihydroorotase reaction [Porter04]. The subunits operate cooperatively during catalysis [Lee05g].

Translation of pyrC is regulated by transcription start site selection. When CTP is abundant, a site distal to the start codon is chosen, allowing formation of a hairpin that blocks ribosome binding. Otherwise, a proximal site is chosen that prevents hairpin formation, allowing translation [Wilson92, Liu94c, Liu94d].

Review: [Turnbough08]

Citations: [Backstrom86]

Locations: cytosol

Map Position: [1,120,784 <- 1,121,830] (24.16 centisomes)
Length: 1047 bp / 348 aa

Molecular Weight of Polypeptide: 38.827 kD (from nucleotide sequence), 38.695 kD (experimental) [Daniel97 ]

Molecular Weight of Multimer: 80.9 kD (experimental) [Washabaugh84]

pI: 4.97 [Washabaugh84]

Unification Links: ASAP:ABE-0003607 , CGSC:329 , DIP:DIP-10624N , EchoBASE:EB0799 , EcoGene:EG10806 , EcoliWiki:b1062 , ModBase:P05020 , OU-Microarray:b1062 , PortEco:pyrC , PR:PRO_000023658 , Pride:P05020 , Protein Model Portal:P05020 , RefSeq:NP_415580 , RegulonDB:EG10806 , SMR:P05020 , String:511145.b1062 , UniProt:P05020

Relationship Links: InterPro:IN-FAMILY:IPR002195 , InterPro:IN-FAMILY:IPR004721 , InterPro:IN-FAMILY:IPR006680 , PDB:Structure:1J79 , PDB:Structure:1XGE , PDB:Structure:2E25 , PDB:Structure:2EG6 , PDB:Structure:2EG7 , PDB:Structure:2EG8 , PDB:Structure:2Z24 , PDB:Structure:2Z25 , PDB:Structure:2Z26 , PDB:Structure:2Z27 , PDB:Structure:2Z28 , PDB:Structure:2Z29 , PDB:Structure:2Z2A , PDB:Structure:2Z2B , Pfam:IN-FAMILY:PF01979 , Prosite:IN-FAMILY:PS00482 , Prosite:IN-FAMILY:PS00483

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0006207 - 'de novo' pyrimidine nucleobase biosynthetic process Inferred from experiment [Jensen84]
GO:0006221 - pyrimidine nucleotide biosynthetic process Inferred by computational analysis [UniProtGOA11a, GOA06]
GO:0019856 - pyrimidine nucleobase biosynthetic process Inferred by computational analysis [GOA01]
GO:0044205 - 'de novo' UMP biosynthetic process Inferred by computational analysis [UniProtGOA12]
Molecular Function: GO:0004151 - dihydroorotase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Washabaugh84]
GO:0008270 - zinc ion binding Inferred from experiment Inferred by computational analysis [GOA06, Washabaugh84]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11a, GOA01]
GO:0016812 - hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in cyclic amides Inferred by computational analysis [GOA01]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05]
GO:0005737 - cytoplasm

MultiFun Terms: metabolism biosynthesis of building blocks nucleotides pyrimidine biosynthesis

Essentiality data for pyrC 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:
Curated 26-Jul-2006 by Shearer A , SRI International
Last-Curated ? 16-Jul-2010 by Keseler I , SRI International


Enzymatic reaction of: dihydroorotase

Synonyms: carbamoylaspartic dehydrase, DHOASE, (S)-Dihydroorotate amidohydrolase, L-5,6-dihydroorotate amidohydrolase

EC Number: 3.5.2.3

(S)-dihydroorotate + H2O <=> N-carbamoyl-L-aspartate + 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. [Washabaugh84]

In Pathways: superpathway of histidine, purine, and pyrimidine biosynthesis , superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis , superpathway of pyrimidine ribonucleotides de novo biosynthesis , UMP biosynthesis

Summary:
The kcat/Km of the reaction in the direction of dihydroorotate formation is 1 x 105 M-1 s-1, and in the direction of carbamoyl aspartate formation is 1.2 x 106 M-1 s-1. pH-rate profiles show that the enzyme must be in a protonated state for optimal dihydroorotate formation, and in an unprotonated state for optimal dihydroorotate hydrolysis [Porter04].

Citations: [Li05f]

Cofactors or Prosthetic Groups: Zn2+ [Washabaugh84]

Cofactor Binding Comment: The enzyme tightly binds one Zn atom per chain which is required for catalytic mechanism. Also binds weakly to two other zincs which are not essential for activity. [Brown91a, Washabaugh86]

Kinetic Parameters:

Substrate
Km (μM)
Citations
(S)-dihydroorotate
75.6
[Washabaugh84]
N-carbamoyl-L-aspartate
1700.0, 4000.0, 15000.0
[Porter04, BRENDA14]
N-carbamoyl-L-aspartate
470.0
[Daniel96]

pH(opt): 5.8 [BRENDA14, Washabaugh84], 8 [BRENDA14, Washabaugh84]


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Link97, UniProt11]
UniProt: Removed.
Chain 2 -> 348
[UniProt09]
UniProt: Dihydroorotase;
Metal-Binding-Site 17
[UniProt10]
UniProt: Zinc 1;
Metal-Binding-Site 19
[UniProt10]
UniProt: Zinc 1;
N6-carboxylysine-Modification 103
[UniProt11a]
UniProt: N6-carboxylysine.
Metal-Binding-Site 103
[UniProt10]
UniProt: Zinc 1; via carbamate group;
Metal-Binding-Site 140
[UniProt10]
UniProt: Zinc 2;
Metal-Binding-Site 178
[UniProt10]
UniProt: Zinc 2;
Metal-Binding-Site 251
[UniProt10]
UniProt: Zinc 1;


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b1062 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10806; 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

Backstrom86: Backstrom D, Sjoberg RM, Lundberg LG (1986). "Nucleotide sequence of the structural gene for dihydroorotase of Escherichia coli K12." Eur J Biochem 160(1);77-82. PMID: 2876892

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

Brown91a: Brown DC, Collins KD (1991). "Dihydroorotase from Escherichia coli. Substitution of Co(II) for the active site Zn(II)." J Biol Chem 1991;266(3);1597-604. PMID: 1671037

Daniel96: Daniel R, Kokel B, Caminade E, Martel A, Le Goffic F (1996). "Assay of Escherichia coli dihydroorotase with enantiomeric substrate: practical preparation of carbamyl L-aspartate and high-performance liquid chromatography analysis of catalysis product." Anal Biochem 239(2);130-5. PMID: 8811890

Daniel97: Daniel R, Caminade E, Martel A, Le Goffic F, Canosa D, Carrascal M, Abian J (1997). "Mass spectrometric determination of the cleavage sites in Escherichia coli dihydroorotase induced by a cysteine-specific reagent." J Biol Chem 272(43);26934-9. PMID: 9341128

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

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."

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

Jensen84: Jensen KF, Larsen JN, Schack L, Sivertsen A (1984). "Studies on the structure and expression of Escherichia coli pyrC, pyrD, and pyrF using the cloned genes." Eur J Biochem 140(2);343-52. PMID: 6370696

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

Kim01g: Kim GJ, Lee DE, Kim HS (2001). "High-level expression and one-step purification of cyclic amidohydrolase family enzymes." Protein Expr Purif 23(1);128-33. PMID: 11570854

Lee05g: Lee M, Chan CW, Mitchell Guss J, Christopherson RI, Maher MJ (2005). "Dihydroorotase from Escherichia coli: loop movement and cooperativity between subunits." J Mol Biol 348(3);523-33. PMID: 15826651

Lee07d: Lee M, Maher MJ, Christopherson RI, Guss JM (2007). "Kinetic and structural analysis of mutant Escherichia coli dihydroorotases: a flexible loop stabilizes the transition state." Biochemistry 46(37);10538-50. PMID: 17711307

Lee07h: Lee M, Maher MJ, Guss JM (2007). "Structure of the T109S mutant of Escherichia coli dihydroorotase complexed with the inhibitor 5-fluoroorotate: catalytic activity is reflected by the crystal form." Acta Crystallogr Sect F Struct Biol Cryst Commun 63(Pt 3);154-61. PMID: 17329804

Lee07i: Lee M, Chan CW, Graham SC, Christopherson RI, Guss JM, Maher MJ (2007). "Structures of ligand-free and inhibitor complexes of dihydroorotase from Escherichia coli: implications for loop movement in inhibitor design." J Mol Biol 370(5);812-25. PMID: 17550785

Li05f: Li Y, Raushel FM (2005). "Inhibitors designed for the active site of dihydroorotase." Bioorg Chem 33(6);470-83. PMID: 16213543

Link97: Link AJ, Robison K, Church GM (1997). "Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12." Electrophoresis 18(8);1259-313. PMID: 9298646

Liu94c: Liu J, Turnbough CL (1994). "Identification of the Shine-Dalgarno sequence required for expression and translational control of the pyrC gene in Escherichia coli K-12." J Bacteriol 176(9);2513-6. PMID: 7909541

Liu94d: Liu J, Turnbough CL (1994). "Effects of transcriptional start site sequence and position on nucleotide-sensitive selection of alternative start sites at the pyrC promoter in Escherichia coli." J Bacteriol 176(10);2938-45. PMID: 7910603

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

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

Porter04: Porter TN, Li Y, Raushel FM (2004). "Mechanism of the dihydroorotase reaction." Biochemistry 43(51);16285-92. PMID: 15610022

Thoden01: Thoden JB, Phillips GN, Neal TM, Raushel FM, Holden HM (2001). "Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center." Biochemistry 40(24);6989-97. PMID: 11401542

Turnbough08: Turnbough CL, Switzer RL (2008). "Regulation of pyrimidine biosynthetic gene expression in bacteria: repression without repressors." Microbiol Mol Biol Rev 72(2);266-300, table of contents. PMID: 18535147

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.

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

UniProt11a: UniProt Consortium (2011). "UniProt version 2011-11 released on 2011-11-22 00:00:00." Database.

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."

Washabaugh84: Washabaugh MW, Collins KD (1984). "Dihydroorotase from Escherichia coli. Purification and characterization." J Biol Chem 259(5);3293-8. PMID: 6142052

Washabaugh86: Washabaugh MW, Collins KD (1986). "Dihydroorotase from Escherichia coli. Sulfhydryl group-metal ion interactions." J Biol Chem 1986;261(13);5920-9. PMID: 2871020

Wilson92: Wilson HR, Archer CD, Liu JK, Turnbough CL (1992). "Translational control of pyrC expression mediated by nucleotide-sensitive selection of transcriptional start sites in Escherichia coli." J Bacteriol 174(2);514-24. PMID: 1345912

Other References Related to Gene Regulation

Bsat99: Bsat N, Helmann JD (1999). "Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo." J Bacteriol 181(14);4299-307. PMID: 10400588

Chai07: Chai S, Song CK, Kim SK, Park JH, Wee S (2007). "Effect of fur on pyrC gene expression." J Microbiol 45(6);583-9. PMID: 18176545

Choi90: Choi KY, Zalkin H (1990). "Regulation of Escherichia coli pyrC by the purine regulon repressor protein." J Bacteriol 172(6);3201-7. PMID: 1971620

Wilson87a: Wilson HR, Chan PT, Turnbough CL (1987). "Nucleotide sequence and expression of the pyrC gene of Escherichia coli K-12." J Bacteriol 169(7);3051-8. PMID: 2885307

Wilson90: Wilson HR, Turnbough CL (1990). "Role of the purine repressor in the regulation of pyrimidine gene expression in Escherichia coli K-12." J Bacteriol 1990;172(6);3208-13. PMID: 1971621


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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 Thu Dec 18, 2014, biocyc13.