EzCatDB: D00675
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DB codeD00675
RLCP classification4.12.642320.562 : Addition
5.201.2781500.563 : Elimination
CATH domainDomain 12.30.40.10 : Urease, subunit C; domain 1
Domain 23.20.20.140 : TIM BarrelCatalytic domain
E.C.3.5.4.1

CATH domainRelated DB codes (homologues)
2.30.40.10 : Urease, subunit C; domain 1D00673,D00801,D00873,M00030,M00225,M00226
3.20.20.140 : TIM BarrelS00231,S00232,M00186,D00673,D00801,D00873,M00030,M00225,M00226

Enzyme Name
UniProtKBKEGG

P25524
Protein nameCytosine deaminaseCytosine deaminase
Isocytosine deaminase
SynonymsEC 3.5.4.1
Cytosine aminohydrolase
RefSeqNP_414871.1 (Protein)
NC_000913.2 (DNA/RNA sequence)
YP_488631.1 (Protein)
NC_007779.1 (DNA/RNA sequence)
PfamPF07969 (Amidohydro_3)
[Graphical view]

KEGG pathways
MAP codePathways
MAP00240Pyrimidine metabolism

UniProtKB:Accession NumberP25524
Entry nameCODA_ECOLI
ActivityCytosine + H(2)O = uracil + NH(3).
SubunitHomotetramer.
Subcellular location
CofactorIron or another divalent metal ion.

Compound table: links to PDB-related databases & PoSSuM

CofactorsSubstratesProductsintermediates
KEGG-idC14818C00380C00001C00106C00014I00151
CompoundFe2+cytosineH2OuracilNH34-Hydroxy-cytosine
Typeheavy metalamide group,amine group,aromatic ring (with nitrogen atoms)H2Oamide group,aromatic ring (with nitrogen atoms)amine group,organic ion
ChEBI29033
16040
15377
17568
16134

PubChem27284
597
962
22247451
1174
222

              
1k6wA01UnboundUnbound UnboundUnboundUnbound
1k70A01UnboundUnbound UnboundUnboundUnbound
1r9xA01UnboundUnbound UnboundUnboundUnbound
1r9yA01UnboundUnbound UnboundUnboundUnbound
1r9zA01UnboundUnbound UnboundUnboundUnbound
1ra0A01UnboundUnbound UnboundUnboundUnbound
1ra5A01UnboundUnbound UnboundUnboundUnbound
1rakA01UnboundUnbound UnboundUnboundUnbound
3g77A01UnboundUnbound UnboundUnboundUnbound
3o7uA01UnboundUnbound UnboundUnboundUnbound
3r0dA01UnboundUnbound UnboundUnboundUnbound
3rn6A01UnboundUnbound UnboundUnboundUnbound
1k6wA02Bound:_FEUnbound UnboundUnboundUnbound
1k70A02Bound:_FEUnbound UnboundUnboundIntermediate-analogue:HPY
1r9xA02Bound:_FEUnbound UnboundUnboundUnbound
1r9yA02Bound:_FEUnbound UnboundUnboundUnbound
1r9zA02Bound:_FEUnbound UnboundUnboundUnbound
1ra0A02Bound:_FEUnbound UnboundUnboundIntermediate-analogue:FPY
1ra5A02Bound:_FEUnbound UnboundUnboundIntermediate-analogue:FPY
1rakA02Bound:_FEUnbound UnboundUnboundIntermediate-analogue:FPY
3g77A02Bound:_FEUnbound UnboundUnboundUnbound
3o7uA02Analogue:_ZNUnbound UnboundUnboundIntermediate-analogue:O7U
3r0dA02Analogue:_ZNUnbound UnboundUnboundUnbound
3rn6A02Analogue:_ZNAnalogue:IGA UnboundUnboundUnbound

Active-site residues
resource
PDB;1k6w, 1k70 & literature [6]
pdbCatalytic residuesCofactor-binding residuescomment
           
1k6wA01 
 
 
1k70A01 
 
 
1r9xA01 
 
 
1r9yA01 
 
 
1r9zA01 
 
 
1ra0A01 
 
 
1ra5A01 
 
 
1rakA01 
 
 
3g77A01 
 
 
3o7uA01 
 
 
3r0dA01 
 
 
3rn6A01 
 
 
1k6wA02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
 
1k70A02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
 
1r9xA02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
mutant D314G
1r9yA02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
mutant D314A
1r9zA02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
mutant D314S
1ra0A02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
mutant D314G
1ra5A02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
mutant D314A
1rakA02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
mutant D314S
3g77A02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
mutant V152A, F316C, D317G
3o7uA02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
 
3r0dA02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
 
3rn6A02GLU 217;HIS 246;ASP 313
HIS 61;HIS 63;HIS 214;ASP 313(Fe2+ binding)
 

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[6]p. 693-694
[12]Scheme 4

references
[1]
PubMed ID1925539
JournalScience
Year1991
Volume252
Pages1278-84
AuthorsWilson DK, Rudolph FB, Quiocho FA
TitleAtomic structure of adenosine deaminase complexed with a transition-state analog: understanding catalysis and immunodeficiency mutations.
[2]
PubMed ID8439534
JournalBiochemistry
Year1993
Volume32
Pages1689-94
AuthorsWilson DK, Quiocho FA
TitleA pre-transition-state mimic of an enzyme: X-ray structure of adenosine deaminase with bound 1-deazaadenosine and zinc-activated water.
[3]
PubMed ID8226944
JournalJ Biol Chem
Year1993
Volume268
Pages24005-11
AuthorsPorter DJ, Austin EA
TitleCytosine deaminase. The roles of divalent metal ions in catalysis.
[4]
PubMed ID9622483
JournalBiochemistry
Year1998
Volume37
Pages8314-24
AuthorsWang Z, Quiocho FA
TitleComplexes of adenosine deaminase with two potent inhibitors: X-ray structures in four independent molecules at pH of maximum activity.
[5]
PubMed ID10669789
JournalBiochim Biophys Acta
Year2000
Volume1476
Pages239-52
AuthorsPorter DJ
TitleEscherichia coli cytosine deaminase: the kinetics and thermodynamics for binding of cytosine to the apoenzyme and the Zn(2+) holoenzyme are similar.
[6]
CommentsX-RAY CRYSTALLOGRAPHY (1.75 ANGSTROMS).
PubMed ID11812140
JournalJ Mol Biol
Year2002
Volume315
Pages687-97
AuthorsIreton GC, McDermott G, Black ME, Stoddard BL
TitleThe structure of Escherichia coli cytosine deaminase.
Related PDB1k6w,1k70
Related UniProtKBP25524
[7]
CommentsX-RAY CRYSTALLOGRAPHY (1.6 ANGSTROMS).
PubMed ID12637534
JournalJ Biol Chem
Year2003
Volume278
Pages19111-7
AuthorsKo TP, Lin JJ, Hu CY, Hsu YH, Wang AH, Liaw SH
TitleCrystal structure of yeast cytosine deaminase. Insights into enzyme mechanism and evolution.
[8]
CommentsX-RAY CRYSTALLOGRAPHY (1.14 ANGSTROMS) IN COMPLEX WITH SUBSTRATE ANALOG AND ZINC IONS, AND SUBUNIT.
PubMed ID12906827
JournalStructure
Year2003
Volume11
Pages961-72
AuthorsIreton GC, Black ME, Stoddard BL
TitleThe 1.14 A crystal structure of yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications for genetic chemotherapy.
[9]
PubMed ID15468078
JournalAngew Chem Int Ed Engl
Year2004
Volume43
Pages5396-9
AuthorsSponer JE, Sanz Miguel PJ, Rodriguez-Santiago L, Erxleben A, Krumm M, Sodupe M, Sponer J, Lippert B
TitleMetal-mediated deamination of cytosine: experiment and DFT calculations.
[10]
PubMed ID15248753
JournalBiochemistry
Year2004
Volume43
Pages8957-64
AuthorsMahan SD, Ireton GC, Stoddard BL, Black ME
TitleAlanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference.
[11]
CommentsX-RAY CRYSTALLOGRAPHY.
PubMed ID15381761
JournalProtein Eng Des Sel
Year2004
Volume17
Pages625-33
AuthorsMahan SD, Ireton GC, Knoeber C, Stoddard BL, Black ME
TitleRandom mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy.
Related PDB1r9x,1r9y,1r9z,1ra0,1ra5,1rak
[12]
PubMed ID21545144
JournalBiochemistry
Year2011
Volume50
Pages5077-85
AuthorsHall RS, Fedorov AA, Xu C, Fedorov EV, Almo SC, Raushel FM
TitleThree-dimensional structure and catalytic mechanism of cytosine deaminase.
Related PDB3o7u
[13]
PubMed ID21604715
JournalBiochemistry
Year2011
Volume50
Pages5555-7
AuthorsHitchcock DS, Fedorov AA, Fedorov EV, Dangott LJ, Almo SC, Raushel FM
TitleRescue of the orphan enzyme isoguanine deaminase.
Related PDB3rn6

comments
This enzyme is homologous to adenosine deaminase (EC 3.5.4.4; S00232 in EzCatDB) and guanine deaminase (EC 3.5.4.3; D00873 in EzCatDB).
Although its catalytic site is similar to those of the homologous enzymes, cofactor ion, Fe2+, is different from the cofactor ion, zinc ion, adopted by the homologous enzymes. However, the reaction mechanism seems to be very similar to those homologous enzymes.
According to the literature [12] and the reaction mechanism of the homologous enzymes(S00232 and D00873 in EzCatDB), this enzyme catalyzes two successive reactions (rather than hydrolysis) as follows:
(A) Addition of water to imine carbon to form a tetrahedral intermediate (I00151).
(A1) Asp313 (or Glu217) acts as a general base to deprotonate the Fe2+-bound water. Here, the positive charge of His246 seems to stabilize the activated water. His246 may assist the catalytic function of Glu217 as well (see [12]).
(A2) The activated water makes a nucleophilic attack on the C4 atom of cytosine, whilst Glu217 acts as a general acid to protonate the N3 atom (protonation site) of the cytosine. This reaction leads to the formation of tetrahedral intermediate at the C4 atom, transforming N3-C4 bond from a double bond to a single bond (I00151).
(B) Elimination of amine group from the intermediate (I00151), forming a carbonyl group.
(B1) Asp313 acts as a general acid to protonate the eliminated amine group, releasing the ammonia.
(B2) Asp313 acts as a general base to deprotonate the hydroxyl group, bound to the Fe2+ ion and His246. (Here, His246 may assist the catalytic function of Asp313 as well.) This reaction leads to the enol form of the product, cytosine. (This may be an E1-like reaction, as amine elimination occurs prior to deprotonation.)

createdupdated
2008-05-302012-10-17


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Funded by BIRD/Japan Science and Technology Corporation (JST) (October 2007 - September 2010)
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