EzCatDB: S00383
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DB codeS00383
RLCP classification1.15.9400.1180 : Hydrolysis
CATH domainDomain 13.40.91.10 : Restriction EndonucleaseCatalytic domain
E.C.3.1.21.4
CSA1cfr


Enzyme Name
UniProtKBKEGG

P56200
Protein nameType-2 restriction enzyme Cfr10Itype II site-specific deoxyribonuclease
type II restriction enzyme
SynonymsR.Cfr10I
EC 3.1.21.4
Type II restriction enzyme Cfr10I
Endonuclease Cfr10I
PfamPF07832 (Bse634I)
[Graphical view]


UniProtKB:Accession NumberP56200
Entry nameT2CX_CITFR
ActivityEndonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.
Subunit
Subcellular location
CofactorBinds 2 magnesium ions per subunit.

Compound table: links to PDB-related databases & PoSSuM

CofactorsSubstratesProducts
KEGG-idC00305C00039C00001C00578C00039
CompoundMagnesiumDNAH2ODNA 5'-phosphateDNA
Typedivalent metal (Ca2+, Mg2+)nucleic acidsH2Onucleic acids,phosphate group/phosphate ionnucleic acids
ChEBI18420

15377


PubChem888

962
22247451


             
1cfrAUnboundUnbound UnboundUnbound

Active-site residues
resource
literature [2]
pdbCatalytic residuesCofactor-binding residues
          
1cfrALYS 190
GLU 71;ASP 134;GLU 204(Mg2+ binding)

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[2]p.181-182
[3]Fig.8, Fig.11, p.12-172
[4]Fig.5, p.13492-134942
[5]Fig.1, p.6

references
[1]
Commentscatalysis
PubMed ID7607482
JournalGene
Year1995
Volume157
Pages157-62
AuthorsJeltsch A, Pleckaityte M, Selent U, Wolfes H, Siksnys V, Pingoud A
TitleEvidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases.
[2]
CommentsX-ray crystallography (2.15 Angstroms)
Medline ID96144841
PubMed ID8568865
JournalJ Mol Biol
Year1996
Volume255
Pages176-86
AuthorsBozic D, Grazulis S, Siksnys V, Huber R
TitleCrystal structure of Citrobacter freundii restriction endonuclease Cfr10I at 2.15 A resolution.
Related PDB1cfr
Related UniProtKBP56200
[3]
Commentscatalysis
PubMed ID9210460
JournalEur J Biochem
Year1997
Volume246
Pages1-22
AuthorsPingoud A, Jeltsch A
TitleRecognition and cleavage of DNA by type-II restriction endonucleases.
[4]
CommentsX-ray crystallography (2.15 Angstroms)
PubMed ID9811827
JournalProc Natl Acad Sci U S A
Year1998
Volume95
Pages13489-94
AuthorsHorton NC, Newberry KJ, Perona JJ
TitleMetal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.
[5]
PubMed ID10739241
JournalProtein Sci
Year2000
Volume9
Pages1-9
AuthorsDall'Acqua W, Carter P
TitleSubstrate-assisted catalysis: molecular basis and biological significance.

comments
This enzyme belongs to the type II restriction endonucleases.
Although literature [1] suggests that this enzyme may not employ substrate assistance in catalysis, which is adopted by other type II restriction endonucleases, it is considered to take a similar catalytic mechanism ([2], [3] & [4]).
According to the paper [3], cleavage of DNA by restriction endonucleases yields 3'-OH and 5'-phosphate ends, where hydrolysis of the phosphodiester bonds by EcoRI and EcoRV occurs with inversion of configuration at the phosphorous atom, suggesting an attack of a water molecule in line with the 3'-OH leaving group. In general, hydrolysis of phosphodiester bonds requires three functional entities as follows [3]:
(1) A general base that activates the attacking nucleophile,
(2) A Lewis acid that stabilizes the extra negative charge in the pentacovalent transition state,
(3) An acid that protonates or stabilizes the leaving group.
The literature [3] also described the two possible catalytic mechanisms, the substrate-assisted catalysis model and the two-metal-ion mechanism, as described in the following paragraph. However, this paper supported the substrate-assisted catalysis model more favorably than the two-metal-ion mechanism.
The substrate-assisted catalysis model: The attacking water molecule is oriented and deprotonated by the next phosphate group 3' to the scissile phosphate. The negative charge of the transition state could be stablized by the Mg2+ ion and the semi-conserved lysine. The metal ion is bound by the two conserved acidc amino acid residues. The 3'-O- leaving group is protonated by a Mg2+-bound water [3].
The two-metal-ion mechanism: A metal ion bound at one site is responsible for charge neutralization at the scissile phosphate. The attacking water is considered to be part of the hydration sphere of a metal ion bound at the second site [3].
The literature [4] suggested another possible mechanism, three-metal ion mechanism for type II restriction endonucleases from the structural data, as follows:
A metal ion at site I ligates through water to the 3'-phosphate. A second inner-sphere water molecule on this metal dissociates to provide the attacking hydroxide ion, and this dissociation is aided by the immediately adjacent lysine residue, coresponding to Lys190 in this enzyme. The metal at site III provides stabilization of the incipient negative charge as the transtion state develops. An inner-sphere water on this metal is located within hydrogen-bonding distance of the leaving 3'-oxygen. Thus, the site III metal is suggested to be operative in lowering the pKa of this water, so that it may dissociate to immediately protonate the leaving anion [4]. The site II metal is purely structural [4].
Crystal structures of these type II endonucleases, EcoRV, EcoRI and PvuII bound to DNA show that the relative positions of the scissile and adjacent 3'-phosphates are conserved. Therefore, the two metal ions bound in site I and site III may have similar functions in each of these enzymes [4].
###
However, more recent paper [5] supported the substrate-assisted mechanism for the related enzymes (type II restriction enzymes), ruling out the two-metal-ion mechanism. Thus, we concluded that this enzyme adopts the substrate-assisted mechanism with only one metal ion for catalysis (see EcoRV; S00404 in EzCatDB).
In addition, the pattern of the active site structure is similar to those of EcoRI, EcoRV, BglI and PvuII (S00403, S00404, S00405, & S00390, respectively in EzCatDB), suggesting a similar catalytic mechanism to those by the enzymes, although the structures with ligand molecules are not available.

createdupdated
2002-09-272009-02-26


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