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Escherichia coli K-12 substr. MG1655 Enzyme: dITP/XTP pyrophosphatase



Gene: rdgB Accession Numbers: G7530 (EcoCyc), b2954, ECK2949

Synonyms: yggV

Regulation Summary Diagram: ?

Subunit composition of dITP/XTP pyrophosphatase = [RdgB]2

Summary:
RdgB is a nucleoside triphosphate pyrophosphatase which hydrolyses deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP), mutagenic products of purine nucleotide deamination. RdgB may therefore act to reduce the abundance of noncanonical purines that can be misincorporated during DNA replication, serving as quality control in the metabolism of DNA precursors [Burgis03, Bradshaw03, Chung02, Kouzminova04, Burgis07]. A suppressor screen identified inosine as the main source of noncanonical nucleotides that are removed by RdgB [Budke10].

RdgB does not exhibit strong activity towards dATP, dCTP, or dTTP; the range of hydrolyzed substrates suggests that the O6 atom of the nucleotide substrate is critical for RdgB activity [Chung02]. Crystal structures of free RdgB and in complexes with the ITP substrate or IMP product have been solved. Substrate binding induces closure of the active site and positioning of conserved amino acid residues for catalysis. Site-directed mutagenesis allowed identification of residues that are essential for catalytic activity, and a mechanism for catalysis and substrate specificity was proposed [Savchenko07].

rdgB was first identified due to its genetic interaction with the recA200 allele [Clyman87]. The major consequence of loss of rdgB appears to be chromosomal fragmentation [Lukas06]. An rdgB mutation leads to increased intrachromosomal recombination, compared to wild type, and causes SOS induction [Clyman87]. Both phenotypes are suppressed by purA expression [Clyman91]. A recA200 rdgB double mutant is inviable at elevated temperatures that are nonpermissive for the recA200 allele; under nonpermissive conditions the double mutant exhibits DNA degradation and a defect in DNA replication, but no defect in protein translation [Clyman87]. A moa rdgB double mutant exhibits hypersensitivity to N-6-hydroxylaminopurine (HAP) that is suppressed by an nfi (endonuclease V) mutation; however, this strain exhibits a high rate of mutation [Burgis03]. A rdgB recF strain is viable, and the lethal chromosomal fragmentation phenotype of a rdgB recBC triple mutant is suppressed by an nfi mutation; taken together, these results indicate that the rdgB mutation results in double-strand DNA breaks due to endonuclease V action, consistent with misincorporation of hypoxanthine and/or xanthine during DNA replication [Bradshaw03, Kouzminova04].

Under anaerobiosis, rdgB expression is repressed by FNR, for which a binding site, not shown in the paper, was identified upstream of the gene [Salmon03].

RdgB: "Rec-dependent growth" [Clyman87]

Gene Citations: [Nonaka06]

Locations: cytosol

Map Position: [3,094,703 -> 3,095,296] (66.7 centisomes)
Length: 594 bp / 197 aa

Molecular Weight of Polypeptide: 21.039 kD (from nucleotide sequence), 25 kD (experimental) [Clyman87 ]

Molecular Weight of Multimer: 40.0 kD (experimental) [Chung02]

Unification Links: ASAP:ABE-0009692 , DIP:DIP-12197N , EchoBASE:EB2807 , EcoGene:EG12982 , EcoliWiki:b2954 , Mint:MINT-1322479 , ModBase:P52061 , OU-Microarray:b2954 , PortEco:rdgB , PR:PRO_000023693 , Pride:P52061 , Protein Model Portal:P52061 , RefSeq:NP_417429 , RegulonDB:G7530 , SMR:P52061 , String:511145.b2954 , UniProt:P52061

Relationship Links: InterPro:IN-FAMILY:IPR002637 , InterPro:IN-FAMILY:IPR020922 , Panther:IN-FAMILY:PTHR11067 , PDB:Structure:1K7K , PDB:Structure:2PYU , PDB:Structure:2Q16 , Pfam:IN-FAMILY:PF01725

Gene-Reaction Schematic: ?

Instance reactions of [a nucleoside triphosphate + H2O → a nucleoside 5'-monophosphate + diphosphate + H+] (3.6.1.19):
i1: CTP + H2O → CMP + diphosphate + H+ (3.6.1.65)

i2: dATP + H2O → dAMP + diphosphate + H+ (3.6.1.19)

i3: dUTP + H2O → dUMP + diphosphate + H+ (3.6.1.19/3.6.1.23)

i4: dGTP + H2O → dGMP + diphosphate + H+ (3.6.1.19)

i5: dITP + H2O → dIMP + diphosphate + H+ (3.6.1.66)

i6: ITP + H2O → IMP + diphosphate + H+ (3.6.1.19)

i7: XTP + H2O → XMP + diphosphate + H+ (3.6.1.66)

i8: ATP + H2O → AMP + diphosphate + H+ (3.6.1.8)

i9: dTTP + H2O → dTMP + diphosphate + H+ (3.6.1.19)

i10: UTP + H2O → UMP + diphosphate + H+ (3.6.1.19)

i11: dCTP + H2O → dCMP + diphosphate + H+ (3.6.1.12/3.6.1.19/3.6.1.65)

GO Terms:

Biological Process: GO:0009143 - nucleoside triphosphate catabolic process Inferred from experiment Inferred by computational analysis [GOA01a, Chung02, Budke10]
GO:0006163 - purine nucleotide metabolic process Inferred by computational analysis [GOA06]
GO:0009117 - nucleotide metabolic process Inferred by computational analysis [UniProtGOA11a]
Molecular Function: GO:0000287 - magnesium ion binding Inferred from experiment [Chung02]
GO:0047429 - nucleoside-triphosphate diphosphatase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, Chung02]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0017111 - nucleoside-triphosphatase activity Inferred by computational analysis [GOA01a]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]

MultiFun Terms: cell processes protection
information transfer DNA related

Essentiality data for rdgB knockouts: ?

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

Credits:
Created 10-Dec-2009 by Keseler I , SRI International
Last-Curated ? 11-Dec-2009 by Keseler I , SRI International


Enzymatic reaction of: inosine triphosphate pyrophosphatase (dITP/XTP pyrophosphatase)

Synonyms: ITP pyrophosphatase

ITP + H2O <=> IMP + diphosphate + 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.

The reaction is physiologically favored in the direction shown.

Cofactors or Prosthetic Groups: Mg2+ [Chung02]

Kinetic Parameters:

Substrate
Km (μM)
Citations
ITP
410.0
[Chung02]

pH(opt): 10-10.5 [Chung02]


Enzymatic reaction of: deoxyinosine triphosphate pyrophosphatase (dITP/XTP pyrophosphatase)

Synonyms: deoxyinosine triphosphate diphosphatase, deoxyinosine triphosphate diphosphohydrolase, dITP pyrophosphatase

EC Number: 3.6.1.66

dITP + H2O <=> dIMP + diphosphate + 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.

The reaction is physiologically favored in the direction shown.

Cofactors or Prosthetic Groups: Mg2+ [Chung02]

Kinetic Parameters:

Substrate
Km (μM)
Citations
dITP
22.0
[Burgis07]

pH(opt): 10-10.5 [Chung02]


Enzymatic reaction of: xanthosine triphosphate pyrophosphatase (dITP/XTP pyrophosphatase)

Synonyms: xanthosine triphosphate diphosphatase, xanthosine triphosphate diphosphohydrolase, XTP pyrophosphatase

EC Number: 3.6.1.66

XTP + H2O <=> XMP + diphosphate + 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.

The reaction is physiologically favored in the direction shown.

Alternative Substrates for XTP: GTP [Burgis07 ] , dHAPTP [Burgis07 ] , dGTP [Burgis07 , Chung02 ] , dUTP [Chung02 ]

Summary:
Activity with dGTP and dUTP was first reported to be 10-12% lower than with XTP [Chung02]. The Km for dHAPTP is 16.5 µM, and the Km for dGTP is 792 µM [Burgis07].

Cofactors or Prosthetic Groups: Mg2+ [Chung02]

Kinetic Parameters:

Substrate
Km (μM)
Citations
XTP
330.0
[Chung02]

pH(opt): 10-10.5 [Chung02]


Sequence Features

Feature Class Location Citations Comment
Protein-Segment 8 -> 13
[UniProt11a]
UniProt: Substrate binding; Sequence Annotation Type: region of interest; Non-Experimental Qualifier: by similarity.
Mutagenesis-Variant 13
[Savchenko07]
A K13A mutation results in complete loss of enzymatic activity.
Metal-Binding-Site 40
[UniProt11a]
UniProt: Magnesium or manganese; Non-Experimental Qualifier: by similarity.
Mutagenesis-Variant 41
[Savchenko07]
An E13A mutation results in complete loss of enzymatic activity.
Mutagenesis-Variant 53
[Savchenko07]
A K53A mutation results in complete loss of enzymatic activity.
Metal-Binding-Site 69
[UniProt11a]
UniProt: Magnesium or manganese; Non-Experimental Qualifier: by similarity.
Protein-Segment 69 -> 70
[UniProt11a]
UniProt: Substrate binding; Sequence Annotation Type: region of interest; Non-Experimental Qualifier: by similarity.
Mutagenesis-Variant 69
[Savchenko07]
A D69A mutation results in complete loss of enzymatic activity.
Amino-Acid-Sites-That-Bind 157
[UniProt11a]
UniProt: Substrate; Non-Experimental Qualifier: by similarity.
Amino-Acid-Sites-That-Bind 177
[UniProt11a]
UniProt: Substrate; Non-Experimental Qualifier: by similarity.
Amino-Acid-Sites-That-Bind 183
[UniProt11a]
UniProt: Substrate; Non-Experimental Qualifier: by similarity.


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
Markus Krummenacker on Tue Oct 14, 1997:
Gene object created from Blattner lab Genbank (v. M52) entry.


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

Bradshaw03: Bradshaw JS, Kuzminov A (2003). "RdgB acts to avoid chromosome fragmentation in Escherichia coli." Mol Microbiol 48(6);1711-25. PMID: 12791149

Budke10: Budke B, Kuzminov A (2010). "Production of clastogenic DNA precursors by the nucleotide metabolism in Escherichia coli." Mol Microbiol 75(1);230-45. PMID: 19943897

Burgis03: Burgis NE, Brucker JJ, Cunningham RP (2003). "Repair system for noncanonical purines in Escherichia coli." J Bacteriol 185(10);3101-10. PMID: 12730170

Burgis07: Burgis NE, Cunningham RP (2007). "Substrate specificity of RdgB protein, a deoxyribonucleoside triphosphate pyrophosphohydrolase." J Biol Chem 282(6);3531-8. PMID: 17090528

Chung02: Chung JH, Park HY, Lee JH, Jang Y (2002). "Identification of the dITP- and XTP-hydrolyzing protein from Escherichia coli." J Biochem Mol Biol 35(4);403-8. PMID: 12297000

Clyman87: Clyman J, Cunningham RP (1987). "Escherichia coli K-12 mutants in which viability is dependent on recA function." J Bacteriol 169(9);4203-10. PMID: 2442140

Clyman91: Clyman J, Cunningham RP (1991). "Suppression of the defects in rdgB mutants of Escherichia coli K-12 by the cloned purA gene." J Bacteriol 173(3);1360-2. PMID: 1991730

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

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

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

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

Kouzminova04: Kouzminova EA, Rotman E, Macomber L, Zhang J, Kuzminov A (2004). "RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation." Proc Natl Acad Sci U S A 101(46);16262-7. PMID: 15531636

Lukas06: Lukas L, Kuzminov A (2006). "Chromosomal fragmentation is the major consequence of the rdgB defect in Escherichia coli." Genetics 172(2);1359-62. PMID: 16322510

Nonaka06: Nonaka G, Blankschien M, Herman C, Gross CA, Rhodius VA (2006). "Regulon and promoter analysis of the E. coli heat-shock factor, sigma32, reveals a multifaceted cellular response to heat stress." Genes Dev 20(13);1776-89. PMID: 16818608

Salmon03: Salmon K, Hung SP, Mekjian K, Baldi P, Hatfield GW, Gunsalus RP (2003). "Global gene expression profiling in Escherichia coli K12. The effects of oxygen availability and FNR." J Biol Chem 278(32);29837-55. PMID: 12754220

Savchenko07: Savchenko A, Proudfoot M, Skarina T, Singer A, Litvinova O, Sanishvili R, Brown G, Chirgadze N, Yakunin AF (2007). "Molecular basis of the antimutagenic activity of the house-cleaning inosine triphosphate pyrophosphatase RdgB from Escherichia coli." J Mol Biol 374(4);1091-103. PMID: 17976651

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."


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 Wed Dec 17, 2014, BIOCYC13A.