EzCatDB: S00390
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DB codeS00390
RLCP classification1.15.9400.1180 : Hydrolysis
CATH domainDomain : PvuII Endonuclease; Chain ACatalytic domain

Enzyme Name

Protein nameType-2 restriction enzyme PvuIItype II site-specific deoxyribonuclease
type II restriction enzyme
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.
Subcellular location
CofactorBinds 2 magnesium ions per subunit.

Compound table: links to PDB-related databases & PoSSuM

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




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
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
[9]Fig.2, Fig.3
[11]Fig1, p.6

CommentsX-ray crystallography
PubMed ID8076590
JournalEMBO J
AuthorsCheng X, Balendiran K, Schildkraut I, Anderson JE
TitleStructure of PvuII endonuclease with cognate DNA.
Related PDB1pvi
CommentsX-ray crystallography (2.4 Angstroms)
PubMed ID7664066
JournalNat Struct Biol
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
PubMed ID7607482
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.
PubMed ID9210460
JournalEur J Biochem
AuthorsPingoud A, Jeltsch A
TitleRecognition and cleavage of DNA by type-II restriction endonucleases.
PubMed ID9325303
JournalJ Biol Chem
AuthorsNastri HG, Evans PD, Walker IH, Riggs PD
TitleCatalytic and DNA binding properties of PvuII restriction endonuclease mutants.
CommentsX-ray crystallography (1.9 Angstroms)
PubMed ID9628337
JournalBiol Chem
AuthorsHorton JR, Bonventre J, Cheng X
TitleHow is modification of the DNA substrate recognized by the PvuII restriction endonuclease?
Related PDB2pvi
CommentsX-ray crystallography (2.15 Angstroms)
PubMed ID9811827
JournalProc Natl Acad Sci U S A
AuthorsHorton NC, Newberry KJ, Perona JJ
TitleMetal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.
PubMed ID9878366
JournalJ Mol Biol
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.
CommentsX-ray crystallography
PubMed ID10903853
JournalJ Mol Biol
AuthorsHorton JR, Cheng X
TitlePvuII endonuclease contains two calcium ions in active sites.
Related PDB1eyu,1f0o
PubMed ID10978180
AuthorsDupureur CM, Conlan LH
TitleA catalytically deficient active site variant of PvuII endonuclease binds Mg(II) ions.
PubMed ID10739241
JournalProtein Sci
AuthorsDall'Acqua W, Carter P
TitleSubstrate-assisted catalysis: molecular basis and biological significance.
PubMed ID11491304
JournalJ Mol Biol
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.
PubMed ID11536360
AuthorsDominguez MA Jr, Thornton KC, Melendez MG, Dupureur CM
TitleDifferential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease.
PubMed ID12445784
JournalJ Mol Biol
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.
PubMed ID12475233
AuthorsConlan LH, Dupureur CM
TitleMultiple metal ions drive DNA association by PvuII endonuclease.

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.)


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