DB code: S00383

RLCP classification 1.15.9400.1180 : Hydrolysis
CATH domain 3.40.91.10 : Restriction Endonuclease Catalytic domain
E.C. 3.1.21.4
CSA 1cfr
M-CSA 1cfr
MACiE

CATH domain Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB Protein name Synonyms Pfam
P56200 Type-2 restriction enzyme Cfr10I
R.Cfr10I
EC 3.1.21.4
Type II restriction enzyme Cfr10I
Endonuclease Cfr10I
PF07832 (Bse634I)
[Graphical View]

KEGG enzyme name
type II site-specific deoxyribonuclease
type II restriction enzyme

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P56200 T2CX_CITFR Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates. Binds 2 magnesium ions per subunit.

KEGG Pathways
Map code Pathways E.C.

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00305 C00039 C00001 C00578 C00039
E.C.
Compound Magnesium DNA H2O DNA 5'-phosphate DNA
Type divalent metal (Ca2+, Mg2+) nucleic acids H2O nucleic acids,phosphate group/phosphate ion nucleic acids
ChEBI 18420
15377
PubChem 888
22247451
962
1cfrA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound

Reference for Active-site residues
resource references E.C.
literature [2]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1cfrA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 190 GLU 71;ASP 134;GLU 204(Mg2+ binding)

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[2]
p.181-182
[3]
Fig.8, Fig.11, p.12-17 2
[4]
Fig.5, p.13492-13494 2
[5]
Fig.1, p.6

References
[1]
Resource
Comments catalysis
Medline ID
PubMed ID 7607482
Journal Gene
Year 1995
Volume 157
Pages 157-62
Authors Jeltsch A, Pleckaityte M, Selent U, Wolfes H, Siksnys V, Pingoud A
Title Evidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases.
Related PDB
Related UniProtKB
[2]
Resource
Comments X-ray crystallography (2.15 Angstroms)
Medline ID 96144841
PubMed ID 8568865
Journal J Mol Biol
Year 1996
Volume 255
Pages 176-86
Authors Bozic D, Grazulis S, Siksnys V, Huber R
Title Crystal structure of Citrobacter freundii restriction endonuclease Cfr10I at 2.15 A resolution.
Related PDB 1cfr
Related UniProtKB P56200
[3]
Resource
Comments catalysis
Medline ID
PubMed ID 9210460
Journal Eur J Biochem
Year 1997
Volume 246
Pages 1-22
Authors Pingoud A, Jeltsch A
Title Recognition and cleavage of DNA by type-II restriction endonucleases.
Related PDB
Related UniProtKB
[4]
Resource
Comments X-ray crystallography (2.15 Angstroms)
Medline ID
PubMed ID 9811827
Journal Proc Natl Acad Sci U S A
Year 1998
Volume 95
Pages 13489-94
Authors Horton NC, Newberry KJ, Perona JJ
Title Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID 10739241
Journal Protein Sci
Year 2000
Volume 9
Pages 1-9
Authors Dall'Acqua W, Carter P
Title Substrate-assisted catalysis: molecular basis and biological significance.
Related PDB
Related UniProtKB

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.

Created Updated
2002-09-27 2009-02-26