EzCatDB: S00390
Related links:    PDB-formatted query search system Fasta-formatted query search system Fasta-formatted query search system

DB codeS00390
RLCP classification1.15.9400.1180 : Hydrolysis
CATH domainDomain 13.40.210.10 : PvuII Endonuclease; Chain ACatalytic domain
E.C.3.1.21.4
CSA1pvi


Enzyme Name
UniProtKBKEGG

P23657
Protein nameType-2 restriction enzyme PvuIItype II site-specific deoxyribonuclease
type II restriction enzyme
SynonymsR.PvuII
EC 3.1.21.4
Type II restriction enzyme PvuII
Endonuclease PvuII
PfamPF09225 (Endonuc-PvuII)
[Graphical view]


UniProtKB:Accession NumberP23657
Entry nameT2P2_PROVU
ActivityEndonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.
SubunitHomodimer.
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


             
1eyuAUnboundBound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain D:double stranded DNA) UnboundUnbound
1eyuBUnboundBound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain C:double stranded DNA) UnboundUnbound
1f0oAAnalogue:2x_CABound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain D:double stranded DNA) UnboundUnbound
1f0oBAnalogue:2x_CABound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain C:double stranded DNA) UnboundUnbound
1pviAUnboundBound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain D:double stranded DNA) UnboundUnbound
1pviBUnboundBound:T-G-A-C-C-A-G-C-T-G-G-T-C(chain C:double stranded DNA) UnboundUnbound
1pvuAUnboundUnbound UnboundUnbound
1pvuBUnboundUnbound UnboundUnbound
2pviAUnboundAnalogue:T-G-A-C-C-A-G-I5C-T-G-G-T-C(chain D:double stranded iodinated cognate DNA) UnboundUnbound
2pviBUnboundAnalogue:T-G-A-C-C-A-G-I5C-T-G-G-T-C(chain C:double stranded iodinated cognate DNA) UnboundUnbound

Active-site residues
resource
PDB;2pvi
pdbCatalytic residuesCofactor-binding residues
          
1eyuALYS 70
ASP 58;GLU 68(two Mg2+ binding)
1eyuBLYS 70
ASP 58;GLU 68(two Mg2+ binding)
1f0oALYS 70
ASP 58;GLU 68(two Mg2+ binding)
1f0oBLYS 70
ASP 58;GLU 68(two Mg2+ binding)
1pviALYS 70
ASP 58;GLU 68(two Mg2+ binding)
1pviBLYS 70
ASP 58;GLU 68(two Mg2+ binding)
1pvuALYS 70
ASP 58;GLU 68(two Mg2+ binding)
1pvuBLYS 70
ASP 58;GLU 68(two Mg2+ binding)
2pviALYS 70
ASP 58;GLU 68(two Mg2+ binding)
2pviBLYS 70
ASP 58;GLU 68(two Mg2+ binding)

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[4]Fig.8, Fig.11, p.12-172
[7]Fig.5, p.13492-134942
[8]p.1496-1498
[9]Fig.2, Fig.3
[11]Fig1, p.6

references
[1]
CommentsX-ray crystallography
PubMed ID8076590
JournalEMBO J
Year1994
Volume13
Pages3927-35
AuthorsCheng X, Balendiran K, Schildkraut I, Anderson JE
TitleStructure of PvuII endonuclease with cognate DNA.
Related PDB1pvi
[2]
CommentsX-ray crystallography (2.4 Angstroms)
PubMed ID7664066
JournalNat Struct Biol
Year1994
Volume1
Pages469-75
AuthorsAthanasiadis A, Vlassi M, Kotsifaki D, Tucker PA, Wilson KS, Kokkinidis M
TitleCrystal structure of PvuII endonuclease reveals extensive structural homologies to EcoRV.
Related PDB1pvu
[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]
Commentscatalysis
PubMed ID9210460
JournalEur J Biochem
Year1997
Volume246
Pages1-22
AuthorsPingoud A, Jeltsch A
TitleRecognition and cleavage of DNA by type-II restriction endonucleases.
[5]
Commentscatalysis
PubMed ID9325303
JournalJ Biol Chem
Year1997
Volume272
Pages25761-7
AuthorsNastri HG, Evans PD, Walker IH, Riggs PD
TitleCatalytic and DNA binding properties of PvuII restriction endonuclease mutants.
[6]
CommentsX-ray crystallography (1.9 Angstroms)
PubMed ID9628337
JournalBiol Chem
Year1998
Volume379
Pages451-8
AuthorsHorton JR, Bonventre J, Cheng X
TitleHow is modification of the DNA substrate recognized by the PvuII restriction endonuclease?
Related PDB2pvi
[7]
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.
[8]
Commentscatalysis
PubMed ID9878366
JournalJ Mol Biol
Year1998
Volume284
Pages1491-504
AuthorsHorton JR, Nastri HG, Riggs PD, Cheng X
TitleAsp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis.
[9]
CommentsX-ray crystallography
PubMed ID10903853
JournalJ Mol Biol
Year2000
Volume300
Pages1049-56
AuthorsHorton JR, Cheng X
TitlePvuII endonuclease contains two calcium ions in active sites.
Related PDB1eyu,1f0o
[10]
Commentscatalysis
PubMed ID10978180
JournalBiochemistry
Year2000
Volume39
Pages10921-7
AuthorsDupureur CM, Conlan LH
TitleA catalytically deficient active site variant of PvuII endonuclease binds Mg(II) ions.
[11]
PubMed ID10739241
JournalProtein Sci
Year2000
Volume9
Pages1-9
AuthorsDall'Acqua W, Carter P
TitleSubstrate-assisted catalysis: molecular basis and biological significance.
[12]
Commentscatalysis
PubMed ID11491304
JournalJ Mol Biol
Year2001
Volume309
Pages89-97
AuthorsSimoncsits A, Tjornhammar ML, Rasko T, Kiss A, Pongor S
TitleCovalent joining of the subunits of a homodimeric type II restriction endonuclease: single-chain PvuII endonuclease.
[13]
Commentscatalysis
PubMed ID11536360
JournalProteins
Year2001
Volume45
Pages55-61
AuthorsDominguez MA Jr, Thornton KC, Melendez MG, Dupureur CM
TitleDifferential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease.
[14]
Commentscatalysis
PubMed ID12445784
JournalJ Mol Biol
Year2002
Volume324
Pages491-500
AuthorsRauch C, Trieb M, Flader W, Wellenzohn B, Winger RH, Mayer E, Hallbrucker A, Liedl KR
TitlePvuII-endonuclease induces structural alterations at the scissile phosphate group of its cognate DNA.
[15]
Commentscatalysis
PubMed ID12475233
JournalBiochemistry
Year2002
Volume41
Pages14848-55
AuthorsConlan LH, Dupureur CM
TitleMultiple metal ions drive DNA association by PvuII endonuclease.

comments
This enzyme belongs to the type II restriction endonucleases.
According to the paper [4], 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 [4]:
(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 [4] 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.
(1) 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 [4].
(2) 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 [4].
The literature [8] described two possible catalytic mechanisms for type II restriction endonucleases. Both mechanisms involve two acidic amino acids (Asp58 and Glu68) and one basic amino acid (Lys70). In the enzyme-DNA complex, a binding site for a Mg2+ ion is formed by the sidechains of Asp58 and Glu68. The amino group of Lys70 stabilizes the transition state and acts as a Lewis acid in the reaction.
(1) Substrate-assisted, one metal ion mechanism: The phosphate group of the adjacent T(+2) should act as a general base where the attacking water molecule "A" is deprotonated.
(2) Two metal ion mechanism: A second Mg2+ ion should be coordinated to Glu55/Asp58 site and would neutralize the charge of the scissile phosphate of C(+1). However, Glu55 is not crucial in the metal coordination; it does not exclude the possiblity that a second Mg2+ could be coordinated in the same vicinity via water molecules, protein mainchain atoms, and/or the DNA phosphate backbone.
The literature [7] also suggested another possible mechanism, three-metal ion mechanism for type II restriction endonucleases from the structural data of EcoRV, 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, corresponding to Lys70 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 [7]. The site II metal is purely structural [7].
Crystal structures of these type II endonucleases, EcoRV, EcoRI and this enzyme, 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 [7].
###
More recently, several papers including [11] supported the substrate-assisted mechanism for this enzyme and 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).
Considering the structure of 1f0o and in-line attack by water on the scissile phosphoric ester bond, the substrate-assisted mechanism seems to be more likely, and the reaction probably proceeds as follows:
(1) Substrate-assisted Water activation by the 3'-phosphate group of adjacent nucleotide of the DNA (distance between the base-phosphate oxygen and the water, 2.36 A, and that between the water and calcium ion, 2.79 A, in enzyme chain B). This activated water is stabilized by lys70 (distance 3.39 A in enzyme chain B).
(2) The activated water makes a nucleophilic attack on the phosphorus atom in line with the P-O3' bond. (distance 3.39 A in enzyme chain B)
(3) Transition-state is stabilized by (Lys70 and) magnesium ion (distance 4.30 A with lys70, and 2.32 A and 2.60 A with the two calcium ions, analogues of magnesium ions, in enzyme chain B)
(4) Another water, which is bound to magnesium ion and Asp58, acts as a general acid to protonate the leaving O3' atom. (There is no catalytic acid in enzyme chain B for DNA chain C, whereas distance between O3' & water 3.17A, that between calcium and water, 3.46 A, and that between Asp58 and water, 2.55 A for DNA chain D and protein chain A) (This water also interacts with the phosphate oxygen (distance 3.39 A) in chain A.)

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 2008)
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)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2012 - March 2013)
Supported by the commission for the Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI) (October 2012 - )
© Biotechnology Research Institute for Drug Discovery, AIST, 2015-2016 All Rights Reserved.
© Computational Biology Research Center, AIST, 2004-2016 All Rights Reserved.