EzCatDB: S00404

DB codeS00404
RLCP classification1.15.9400.1180
CATH domainDomain 13.40.600.10Catalytic domain
E.C.3.1.21.4


Enzyme Name
Swiss-protKEGG

P04390
Protein nameType-2 restriction enzyme EcoRVtype II site-specific deoxyribonuclease
type II restriction enzyme
SynonymsR.EcoRV
EC 3.1.21.4
Type II restriction enzyme EcoRV
Endonuclease EcoRV


Swiss-prot:Accession NumberP04390
Entry nameT2E5_ECOLX
ActivityEndonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.
SubunitHomodimer.
Subcellular location
CofactorBinds 2 magnesium ions per subunit.


CofactorsSubstratesProducts
KEGG-idC00305C00039C00001C00578C00039
CompoundMagnesiumDNAH2ODNA 5'-phosphateDNA
Typedivalent metal (Ca2+, Mg2+)nucleic acidsH2Onucleic acids,phosphate group/phosphate ionnucleic acids
1az0AAnalogue:_CABound:A-A-A-G-A-T-A-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1az0BAnalogue:_CABound:A-A-A-G-A-T-A-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1az3AUnboundUnbound
UnboundUnbound
1az3BUnboundUnbound
UnboundUnbound
1az4AUnboundUnbound
UnboundUnbound
1az4BUnboundUnbound
UnboundUnbound
1b94AAnalogue:_CABound:A-A-A-G-A-T-A-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1b94BAnalogue:_CABound:A-A-A-G-A-T-A-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1b95AUnboundBound:A-A-A-G-A-T-A-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1b95BUnboundBound:A-A-A-G-A-T-A-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1b96AUnboundBound:A-A-A-G-A-T-A-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1b96BUnboundBound:A-A-A-G-A-T-A-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1b97AUnboundBound:A-A-A-G-A-T-A-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1b97BUnboundBound:A-A-A-G-A-T-A-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1bgbAUnboundBound:G-G-G-A-T-A-T-C-C-C(chain D:double stranded DNA)
UnboundUnbound
1bgbBUnboundBound:C-G-G-G-A-T-A-T-C-C-C(chain C:double stranded DNA)
UnboundUnbound
1bssAAnalogue:2x_CABound:A-A-A-G-A-T-A-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1bssBUnboundBound:A-A-A-G-A-T-A-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1bsuAAnalogue:_CAAnalogue:A-A-G-A-5CM-I-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1bsuBAnalogue:_CAAnalogue:A-A-A-G-A-5CM-I-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1buaAUnboundAnalogue:A-A-A-G-A-C-I-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1buaBUnboundAnalogue:A-A-A-G-A-C-I-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1eo3ABound:2x_MGAnalogue:A-A-G-A-TSP-A-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1eo3BBound:2x_MGAnalogue:C-A-A-G-A-TSP-A-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1eo4AAnalogue:4x_MNAnalogue:A-A-G-A-TSP-A-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1eo4BAnalogue:_MNAnalogue:C-A-A-G-A-TSP-A-T-C-T(chain D:double stranded DNA)
UnboundUnbound
1eonAAnalogue:2x_CLAnalogue:A-A-A-G-A-TSP-A-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1eonBAnalogue:2x_CLAnalogue:C-A-A-G-A-TSP-A-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1eooAUnboundBound:G-A-A-G-A-T-A-T-C-T-T-C(chain C:double stranded DNA)
UnboundUnbound
1eooBUnboundBound:G-A-A-G-A-T-A-T-C-T-T-C(chain D:double stranded DNA)
UnboundUnbound
1eopAUnboundBound:A-A-G-A-T-A-T-C-T-T-A(chain C:double stranded DNA)
UnboundUnbound
1eopBUnboundBound:A-A-G-A-T-A-T-C-T-T-A(chain D:double stranded DNA)
UnboundUnbound
1rv5AUnboundUnbound
Analogue:A-T-C-T-T(chain C:cleaved DNA)Bound:A-A-A-G-A-T(chain C:cleaved DNA)
1rv5BUnboundUnbound
Analogue:A-T-C-T-T(chain D:cleaved DNA)Bound:A-A-A-G-A-T(chain D:cleaved DNA)
1rvaAUnboundBound:A-A-A-G-A-T-A-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1rvaBUnboundBound:A-A-A-G-A-T-A-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1rvbAUnboundBound:A-A-A-G-A-T-A-T-C-T-T(chain C:double stranded DNA)
UnboundUnbound
1rvbBBound:2x_MGBound:A-A-A-G-A-T-A-T-C-T-T(chain D:double stranded DNA)
UnboundUnbound
1rvcABound:2x_MGUnbound
Bound:A-T-C-T-T(chain D)Bound:A-A-A-G-A-T(chain C)
1rvcBBound:2x_MGUnbound
Bound:A-T-C-T-T(chain F)Bound:A-A-A-G-A-T(chain E)
1rveAUnboundUnbound
UnboundUnbound
1rveBUnboundUnbound
UnboundUnbound
2rveAUnboundUnbound
Analogue:C-G-A-G-C-T-C-G(chain C)Analogue:C-G-A-G-C-T-C-G(chain F)
2rveBUnboundUnbound
Analogue:C-G-A-G-C-T-C-G(chain E)Analogue:C-G-A-G-C-T-C-G(chain D)
4rveAUnboundBound:G-G-G-A-T-A-T-C-C-C(chain E:double stranded DNA)
UnboundUnbound
4rveBUnboundBound:G-G-G-A-T-A-T-C-C-C(chain D:double stranded DNA)
UnboundUnbound
4rveCUnboundBound:G-G-G-A-T-A-T-C-C-C(chain F:single stranded DNA)
UnboundUnbound

Active-site residues
resource
Swiss-prot;P04390
pdbCatalytic residuesCofactor-binding residuescomment
1az0ALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1az0BLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1az3ALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1az3BLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1az4ALYS 92
ASP 74;ASP 90(two Mg2+ binding)
mutant T93A
1az4BLYS 92
ASP 74;ASP 90(two Mg2+ binding)
mutant T93A
1b94ALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1b94BLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1b95ALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1b95BLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1b96ALYS 92
ASP 74;ASP 90(two Mg2+ binding)
mutant Q69E
1b96BLYS 92
ASP 74;ASP 90(two Mg2+ binding)
mutant Q69E
1b97ALYS 92
ASP 74;ASP 90(two Mg2+ binding)
mutant Q69L
1b97BLYS 92
ASP 74;ASP 90(two Mg2+ binding)
mutant Q69L
1bgbALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1bgbBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1bssALYS 92
ASP 74;ASP 90(two Mg2+ binding)
mutant T93A
1bssBLYS 92
ASP 74;ASP 90(two Mg2+ binding)
mutant T93A
1bsuALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1bsuBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1buaALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1buaBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1eo3ALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1eo3BLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1eo4ALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1eo4BLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1eonALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1eonBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1eooALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1eooBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1eopALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1eopBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1rv5ALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1rv5BLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1rvaALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1rvaBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1rvbALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1rvbBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1rvcALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1rvcBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

1rveALYS 92
ASP 74;ASP 90(two Mg2+ binding)

1rveBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

2rveALYS 92
ASP 74;ASP 90(two Mg2+ binding)

2rveBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

4rveALYS 92
ASP 74;ASP 90(two Mg2+ binding)

4rveBLYS 92
ASP 74;ASP 90(two Mg2+ binding)

4rveCLYS 92
ASP 74;ASP 90(two Mg2+ binding)


References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[5]Fig.8, Fig.11, p.12-172
[6]Fig.1, p.11397-11401
[10]Fig.5, p.13492-134942
[12]Fig.6, p.6583-6585
[15]Fig1, p.6

references
[1]
CommentsX-ray crystallography (3.0 Angstroms)
Medline ID93259119
PubMed ID8491171
JournalEMBO J
Year1993
Volume12
Pages1781-95
AuthorsWinkler FK, Banner DW, Oefner C, Tsernoglou D, Brown RS, Heathman SP, Bryan RK, Martin PD, Petratos K, Wilson KS
TitleThe crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.
Related PDB1rve,2rve,4rve
Related Swiss-protP04390
[2]
CommentsX-ray crystallography (2 Angstroms)
PubMed ID7819264
JournalBiochemistry
Year1995
Volume34
Pages683-96
AuthorsKostrewa D, Winkler FK
TitleMg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution.
Related PDB1rva,1rvb,1rvc
[3]
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.
[4]
CommentsX-ray crystallography (2.4 Angstroms)
Medline ID98035052
PubMed ID9367757
JournalJ Mol Biol
Year1997
Volume273
Pages207-25
AuthorsPerona JJ, Martin AM
TitleConformational transitions and structural deformability of EcoRV endonuclease revealed by crystallographic analysis.
[5]
Commentscatalysis
PubMed ID9210460
JournalEur J Biochem
Year1997
Volume246
Pages1-22
AuthorsPingoud A, Jeltsch A
TitleRecognition and cleavage of DNA by type-II restriction endonucleases.
[6]
PubMed ID9298958
JournalBiochemistry
Year1997
Volume36
Pages11389-401
AuthorsGroll DH, Jeltsch A, Selent U, Pingoud A
TitleDoes the restriction endonuclease EcoRV employ a two-metal-Ion mechanism for DNA cleavage?
[7]
Commentscatalysis
PubMed ID9548954
JournalBiochemistry
Year1998
Volume37
Pages5682-8
AuthorsStahl F, Wende W, Wenz C, Jeltsch A, Pingoud A
TitleIntra- 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.
[8]
CommentsX-ray crystallography (2.1 Angstroms)
Medline ID98371008
PubMed ID9705308
JournalJ Biol Chem
Year1998
Volume273
Pages21721-9
AuthorsHorton NC, Perona JJ
TitleRecognition of flanking DNA sequences by EcoRV endonuclease involves alternative patterns of water-mediated contacts.
Related PDB1bgb
Related Swiss-protP04390
[9]
CommentsX-ray crystallography (2.1 Angstroms)
Medline ID98213664
PubMed ID9545372
JournalJ Mol Biol
Year1998
Volume277
Pages779-87
AuthorsHorton NC, Perona JJ
TitleRole of protein-induced bending in the specificity of DNA recognition: crystal structure of EcoRV endonuclease complexed with d(AAAGAT) + d(ATCTT).
Related PDB1rv5
Related Swiss-protP04390
[10]
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.
Related PDB1bss
[11]
Commentscatalysis
PubMed ID9628339
JournalBiol Chem
Year1998
Volume379
Pages467-73
AuthorsStahl F, Wende W, Jeltsch A, Pingoud A
TitleThe 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.
[12]
Commentscatalysis
PubMed ID10350476
JournalBiochemistry
Year1999
Volume38
Pages6576-86
AuthorsSam MD, Perona JJ
TitleCatalytic roles of divalent metal ions in phosphoryl transfer by EcoRV endonuclease.
[13]
CommentsX-ray crystallography (2.3 Angstroms)
Medline ID99377171
PubMed ID10446231
JournalNucleic Acids Res
Year1999
Volume27
Pages3438-45
AuthorsThomas MP, Brady RL, Halford SE, Sessions RB, Baldwin GS
TitleStructural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.
Related PDB1b94,1b95,1b96,1b97
Related Swiss-protP04390
[14]
CommentsX-ray crystallography
PubMed ID10801972
JournalProc Natl Acad Sci U S A
Year2000
Volume97
Pages5729-34
AuthorsHorton NC, Perona JJ
TitleCrystallographic snapshots along a protein-induced DNA-bending pathway.
Related PDB1eoo,1eop
[15]
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.
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.

createdupdated
2002-09-272009-02-26
Copyright: Nozomi Nagano, JST & CBRC-AIST
Funded by PRESTO/Japan Science and Technology Corporation (JST) (December 2001 - November 2004)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2006)
Funded by Grant-in-Aid for Scientific Research (B)/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (September 2005 - September 2010)
Funded by BIRD/Japan Science and Technology Corporation (JST) (October 2007 - September 2010)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2011 - March 2012)

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