DB code: S00404

RLCP classification 1.15.9400.1180 : Hydrolysis
CATH domain 3.40.600.10 : ECO RV Endonuclease; Chain A Catalytic domain
E.C. 3.1.21.4
CSA
M-CSA
MACiE

CATH domain Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB Protein name Synonyms RefSeq Pfam
P04390 Type-2 restriction enzyme EcoRV
R.EcoRV
EC 3.1.21.4
Type II restriction enzyme EcoRV
Endonuclease EcoRV
NP_863580.1 (Protein)
NC_005019.1 (DNA/RNA sequence)
YP_007316617.1 (Protein)
NC_019982.1 (DNA/RNA sequence)
PF09233 (Endonuc-EcoRV)
[Graphical View]

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

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P04390 T2E5_ECOLX Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates. Homodimer. 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
 18420
 		15377
 15377
PubChem 888
 888
 		22247451
 962
 22247451
 962
1az0A Analogue:_CA Bound:A-A-A-G-A-T-A-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1az0B Analogue:_CA Bound:A-A-A-G-A-T-A-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1az3A Unbound Unbound Unbound Unbound
1az3B Unbound Unbound Unbound Unbound
1az4A Unbound Unbound Unbound Unbound
1az4B Unbound Unbound Unbound Unbound
1b94A Analogue:_CA Bound:A-A-A-G-A-T-A-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1b94B Analogue:_CA Bound:A-A-A-G-A-T-A-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1b95A Unbound Bound:A-A-A-G-A-T-A-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1b95B Unbound Bound:A-A-A-G-A-T-A-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1b96A Unbound Bound:A-A-A-G-A-T-A-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1b96B Unbound Bound:A-A-A-G-A-T-A-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1b97A Unbound Bound:A-A-A-G-A-T-A-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1b97B Unbound Bound:A-A-A-G-A-T-A-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1bgbA Unbound Bound:G-G-G-A-T-A-T-C-C-C (chain D:double stranded DNA) Unbound Unbound
1bgbB Unbound Bound:C-G-G-G-A-T-A-T-C-C-C (chain C:double stranded DNA) Unbound Unbound
1bssA Analogue:2x_CA Bound:A-A-A-G-A-T-A-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1bssB Unbound Bound:A-A-A-G-A-T-A-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1bsuA Analogue:_CA Analogue:A-A-G-A-5CM-I-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1bsuB Analogue:_CA Analogue:A-A-A-G-A-5CM-I-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1buaA Unbound Analogue:A-A-A-G-A-C-I-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1buaB Unbound Analogue:A-A-A-G-A-C-I-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1eo3A Bound:2x_MG Analogue:A-A-G-A-TSP-A-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1eo3B Bound:2x_MG Analogue:C-A-A-G-A-TSP-A-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1eo4A Analogue:4x_MN Analogue:A-A-G-A-TSP-A-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1eo4B Analogue:_MN Analogue:C-A-A-G-A-TSP-A-T-C-T (chain D:double stranded DNA) Unbound Unbound
1eonA Analogue:2x_CL Analogue:A-A-A-G-A-TSP-A-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1eonB Analogue:2x_CL Analogue:C-A-A-G-A-TSP-A-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1eooA Unbound Bound:G-A-A-G-A-T-A-T-C-T-T-C (chain C:double stranded DNA) Unbound Unbound
1eooB Unbound Bound:G-A-A-G-A-T-A-T-C-T-T-C (chain D:double stranded DNA) Unbound Unbound
1eopA Unbound Bound:A-A-G-A-T-A-T-C-T-T-A (chain C:double stranded DNA) Unbound Unbound
1eopB Unbound Bound:A-A-G-A-T-A-T-C-T-T-A (chain D:double stranded DNA) Unbound Unbound
1rv5A Unbound Unbound Analogue:A-T-C-T-T (chain C:cleaved DNA) Bound:A-A-A-G-A-T (chain C:cleaved DNA)
1rv5B Unbound Unbound Analogue:A-T-C-T-T (chain D:cleaved DNA) Bound:A-A-A-G-A-T (chain D:cleaved DNA)
1rvaA Unbound Bound:A-A-A-G-A-T-A-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1rvaB Unbound Bound:A-A-A-G-A-T-A-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1rvbA Unbound Bound:A-A-A-G-A-T-A-T-C-T-T (chain C:double stranded DNA) Unbound Unbound
1rvbB Bound:2x_MG Bound:A-A-A-G-A-T-A-T-C-T-T (chain D:double stranded DNA) Unbound Unbound
1rvcA Bound:2x_MG Unbound Bound:A-T-C-T-T (chain D) Bound:A-A-A-G-A-T (chain C)
1rvcB Bound:2x_MG Unbound Bound:A-T-C-T-T (chain F) Bound:A-A-A-G-A-T (chain E)
1rveA Unbound Unbound Unbound Unbound
1rveB Unbound Unbound Unbound Unbound
2rveA Unbound Unbound Analogue:C-G-A-G-C-T-C-G (chain C) Analogue:C-G-A-G-C-T-C-G (chain F)
2rveB Unbound Unbound Analogue:C-G-A-G-C-T-C-G (chain E) Analogue:C-G-A-G-C-T-C-G (chain D)
4rveA Unbound Bound:G-G-G-A-T-A-T-C-C-C (chain E:double stranded DNA) Unbound Unbound
4rveB Unbound Bound:G-G-G-A-T-A-T-C-C-C (chain D:double stranded DNA) Unbound Unbound
4rveC Unbound Bound:G-G-G-A-T-A-T-C-C-C (chain F:single stranded DNA) Unbound Unbound

Reference for Active-site residues
resource references E.C.
Swiss-prot;P04390

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1az0A LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1az0B LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1az3A LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1az3B LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1az4A LYS 92 ASP 74;ASP 90(two Mg2+ binding) mutant T93A
1az4B LYS 92 ASP 74;ASP 90(two Mg2+ binding) mutant T93A
1b94A LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1b94B LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1b95A LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1b95B LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1b96A LYS 92 ASP 74;ASP 90(two Mg2+ binding) mutant Q69E
1b96B LYS 92 ASP 74;ASP 90(two Mg2+ binding) mutant Q69E
1b97A LYS 92 ASP 74;ASP 90(two Mg2+ binding) mutant Q69L
1b97B LYS 92 ASP 74;ASP 90(two Mg2+ binding) mutant Q69L
1bgbA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1bgbB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1bssA LYS 92 ASP 74;ASP 90(two Mg2+ binding) mutant T93A
1bssB LYS 92 ASP 74;ASP 90(two Mg2+ binding) mutant T93A
1bsuA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1bsuB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1buaA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1buaB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1eo3A LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1eo3B LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1eo4A LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1eo4B LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1eonA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1eonB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1eooA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1eooB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1eopA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1eopB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1rv5A LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1rv5B LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1rvaA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1rvaB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1rvbA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1rvbB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1rvcA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1rvcB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1rveA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
1rveB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
2rveA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
2rveB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
4rveA LYS 92 ASP 74;ASP 90(two Mg2+ binding)
4rveB LYS 92 ASP 74;ASP 90(two Mg2+ binding)
4rveC LYS 92 ASP 74;ASP 90(two Mg2+ binding)

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[5]
Fig.8, Fig.11, p.12-17 2
[6]
Fig.1, p.11397-11401
[10]
Fig.5, p.13492-13494 2
[12]
Fig.6, p.6583-6585
[15]
Fig1, p.6

References
[1]
Resource
Comments X-ray crystallography (3.0 Angstroms)
Medline ID 93259119
PubMed ID 8491171
Journal EMBO J
Year 1993
Volume 12
Pages 1781-95
Authors Winkler FK, Banner DW, Oefner C, Tsernoglou D, Brown RS, Heathman SP, Bryan RK, Martin PD, Petratos K, Wilson KS
Title The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.
Related PDB 1rve 2rve 4rve
Related UniProtKB P04390
[2]
Resource
Comments X-ray crystallography (2 Angstroms)
Medline ID
PubMed ID 7819264
Journal Biochemistry
Year 1995
Volume 34
Pages 683-96
Authors Kostrewa D, Winkler FK
Title Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution.
Related PDB 1rva 1rvb 1rvc
Related UniProtKB
[3]
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
[4]
Resource
Comments X-ray crystallography (2.4 Angstroms)
Medline ID 98035052
PubMed ID 9367757
Journal J Mol Biol
Year 1997
Volume 273
Pages 207-25
Authors Perona JJ, Martin AM
Title Conformational transitions and structural deformability of EcoRV endonuclease revealed by crystallographic analysis.
Related PDB
Related UniProtKB
[5]
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
[6]
Resource
Comments
Medline ID
PubMed ID 9298958
Journal Biochemistry
Year 1997
Volume 36
Pages 11389-401
Authors Groll DH, Jeltsch A, Selent U, Pingoud A
Title Does the restriction endonuclease EcoRV employ a two-metal-Ion mechanism for DNA cleavage?
Related PDB
Related UniProtKB
[7]
Resource
Comments catalysis
Medline ID
PubMed ID 9548954
Journal Biochemistry
Year 1998
Volume 37
Pages 5682-8
Authors Stahl F, Wende W, Wenz C, Jeltsch A, Pingoud A
Title Intra- vs intersubunit communication in the homodimeric restriction enzyme EcoRV: Thr 37 and Lys 38 involved in indirect readout are only important for the catalytic activity of their own subunit.
Related PDB
Related UniProtKB
[8]
Resource
Comments X-ray crystallography (2.1 Angstroms)
Medline ID 98371008
PubMed ID 9705308
Journal J Biol Chem
Year 1998
Volume 273
Pages 21721-9
Authors Horton NC, Perona JJ
Title Recognition of flanking DNA sequences by EcoRV endonuclease involves alternative patterns of water-mediated contacts.
Related PDB 1bgb
Related UniProtKB P04390
[9]
Resource
Comments X-ray crystallography (2.1 Angstroms)
Medline ID 98213664
PubMed ID 9545372
Journal J Mol Biol
Year 1998
Volume 277
Pages 779-87
Authors Horton NC, Perona JJ
Title Role of protein-induced bending in the specificity of DNA recognition: crystal structure of EcoRV endonuclease complexed with d(AAAGAT) + d(ATCTT).
Related PDB 1rv5
Related UniProtKB P04390
[10]
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 1bss
Related UniProtKB
[11]
Resource
Comments catalysis
Medline ID
PubMed ID 9628339
Journal Biol Chem
Year 1998
Volume 379
Pages 467-73
Authors Stahl F, Wende W, Jeltsch A, Pingoud A
Title The mechanism of DNA cleavage by the type II restriction enzyme EcoRV: Asp36 is not directly involved in DNA cleavage but serves to couple indirect readout to catalysis.
Related PDB
Related UniProtKB
[12]
Resource
Comments catalysis
Medline ID
PubMed ID 10350476
Journal Biochemistry
Year 1999
Volume 38
Pages 6576-86
Authors Sam MD, Perona JJ
Title Catalytic roles of divalent metal ions in phosphoryl transfer by EcoRV endonuclease.
Related PDB
Related UniProtKB
[13]
Resource
Comments X-ray crystallography (2.3 Angstroms)
Medline ID 99377171
PubMed ID 10446231
Journal Nucleic Acids Res
Year 1999
Volume 27
Pages 3438-45
Authors Thomas MP, Brady RL, Halford SE, Sessions RB, Baldwin GS
Title Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.
Related PDB 1b94 1b95 1b96 1b97
Related UniProtKB P04390
[14]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 10801972
Journal Proc Natl Acad Sci U S A
Year 2000
Volume 97
Pages 5729-34
Authors Horton NC, Perona JJ
Title Crystallographic snapshots along a protein-induced DNA-bending pathway.
Related PDB 1eoo 1eop
Related UniProtKB
[15]
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.
According to the paper [5], 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 [5]:
(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 [5] 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 [5].
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 [5].
The literature [10] suggests a 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 Lys92. 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 [10]. The site II metal is purely structural [10].
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 [10].
The literature [12] also supports the metal-ion mediated DNA cleavage. The mechanism involves general acid catalysis for the nucleophilic attack of hydroxide ion on the scissile phosphate, and general acid catalysis for protonation of the leaving 3'-O anion. The ionization of two distinct metal-ligated waters respectively generate the attacking hydroxide ion and the proton for donation to the leaving group [12].
###
According to the literature [6] & [11], the second magnesium ion bound to Glu45 is not involved in catalysis, which ruled out the two-metal-ion mechanism, supporting the substrate-assisted mechanism. However, the second metal might be involved in specific DNA binding.
More recent study [15] focused on substrate-assisted mechanism for various enzymes, including this enzyme. Although the acidity of the substrate phosphate (pKa = 2) makes it poorly suited to the proposed role as a general base to deprotonate a water, the protein environment might perturb the pKa of the substrate phosphate significantly (see [15]).
Moreover, considering the structure of 1f0o (of PvuII; S00390 in EzCatDB) and in-line attack by water on the scissile phosphoric ester bond, the substrate-assisted mechanism is more likely.
Taken together, we concluded that the substrate-assisted mechanism with only one metal should be adopted for this enzyme. The reaction probably proceeds as follows:
(1) Substrate-assisted water activation by the 3'-phosphate group of adjacent nucleotide of the DNA. This activated water is stabilized by lys92.
(2) The activated water makes a nucleophilic attack on the phosphorus atom in line with the P-O3' bond.
(3) Transition-state is stabilized by (Lys92 and) magnesium ion.
(4) Another water, which is bound to magnesium ion and Asp74 and Asp90, acts as a general acid to protonate the leaving O3' atom.

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