DB code: S00390

RLCP classification 1.15.9400.1180 : Hydrolysis
CATH domain 3.40.210.10 : PvuII Endonuclease; Chain A Catalytic domain
E.C. 3.1.21.4
CSA 1pvi
M-CSA 1pvi
MACiE

CATH domain Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB Protein name Synonyms Pfam
P23657 Type-2 restriction enzyme PvuII
R.PvuII
EC 3.1.21.4
Type II restriction enzyme PvuII
Endonuclease PvuII
PF09225 (Endonuc-PvuII)
[Graphical View]

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

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P23657 T2P2_PROVU 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
15377
PubChem 888
22247451
962
1eyuA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-G-A-C-C-A-G-C-T-G-G-T-C (chain D:double stranded DNA) Unbound Unbound
1eyuB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-G-A-C-C-A-G-C-T-G-G-T-C (chain C:double stranded DNA) Unbound Unbound
1f0oA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:2x_CA Bound:T-G-A-C-C-A-G-C-T-G-G-T-C (chain D:double stranded DNA) Unbound Unbound
1f0oB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:2x_CA Bound:T-G-A-C-C-A-G-C-T-G-G-T-C (chain C:double stranded DNA) Unbound Unbound
1pviA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-G-A-C-C-A-G-C-T-G-G-T-C (chain D:double stranded DNA) Unbound Unbound
1pviB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-G-A-C-C-A-G-C-T-G-G-T-C (chain C:double stranded DNA) Unbound Unbound
1pvuA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound
1pvuB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound
2pviA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Analogue:T-G-A-C-C-A-G-I5C-T-G-G-T-C (chain D:double stranded iodinated cognate DNA) Unbound Unbound
2pviB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Analogue:T-G-A-C-C-A-G-I5C-T-G-G-T-C (chain C:double stranded iodinated cognate DNA) Unbound Unbound

Reference for Active-site residues
resource references E.C.
PDB;2pvi

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1eyuA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 70 ASP 58;GLU 68(two Mg2+ binding)
1eyuB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 70 ASP 58;GLU 68(two Mg2+ binding)
1f0oA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 70 ASP 58;GLU 68(two Mg2+ binding)
1f0oB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 70 ASP 58;GLU 68(two Mg2+ binding)
1pviA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 70 ASP 58;GLU 68(two Mg2+ binding)
1pviB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 70 ASP 58;GLU 68(two Mg2+ binding)
1pvuA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 70 ASP 58;GLU 68(two Mg2+ binding)
1pvuB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 70 ASP 58;GLU 68(two Mg2+ binding)
2pviA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 70 ASP 58;GLU 68(two Mg2+ binding)
2pviB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 70 ASP 58;GLU 68(two Mg2+ binding)

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[4]
Fig.8, Fig.11, p.12-17 2
[7]
Fig.5, p.13492-13494 2
[8]
p.1496-1498
[9]
Fig.2, Fig.3
[11]
Fig1, p.6

References
[1]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 8076590
Journal EMBO J
Year 1994
Volume 13
Pages 3927-35
Authors Cheng X, Balendiran K, Schildkraut I, Anderson JE
Title Structure of PvuII endonuclease with cognate DNA.
Related PDB 1pvi
Related UniProtKB
[2]
Resource
Comments X-ray crystallography (2.4 Angstroms)
Medline ID
PubMed ID 7664066
Journal Nat Struct Biol
Year 1994
Volume 1
Pages 469-75
Authors Athanasiadis A, Vlassi M, Kotsifaki D, Tucker PA, Wilson KS, Kokkinidis M
Title Crystal structure of PvuII endonuclease reveals extensive structural homologies to EcoRV.
Related PDB 1pvu
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 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
[5]
Resource
Comments catalysis
Medline ID
PubMed ID 9325303
Journal J Biol Chem
Year 1997
Volume 272
Pages 25761-7
Authors Nastri HG, Evans PD, Walker IH, Riggs PD
Title Catalytic and DNA binding properties of PvuII restriction endonuclease mutants.
Related PDB
Related UniProtKB
[6]
Resource
Comments X-ray crystallography (1.9 Angstroms)
Medline ID
PubMed ID 9628337
Journal Biol Chem
Year 1998
Volume 379
Pages 451-8
Authors Horton JR, Bonventre J, Cheng X
Title How is modification of the DNA substrate recognized by the PvuII restriction endonuclease?
Related PDB 2pvi
Related UniProtKB
[7]
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
[8]
Resource
Comments catalysis
Medline ID
PubMed ID 9878366
Journal J Mol Biol
Year 1998
Volume 284
Pages 1491-504
Authors Horton JR, Nastri HG, Riggs PD, Cheng X
Title Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis.
Related PDB
Related UniProtKB
[9]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 10903853
Journal J Mol Biol
Year 2000
Volume 300
Pages 1049-56
Authors Horton JR, Cheng X
Title PvuII endonuclease contains two calcium ions in active sites.
Related PDB 1eyu 1f0o
Related UniProtKB
[10]
Resource
Comments catalysis
Medline ID
PubMed ID 10978180
Journal Biochemistry
Year 2000
Volume 39
Pages 10921-7
Authors Dupureur CM, Conlan LH
Title A catalytically deficient active site variant of PvuII endonuclease binds Mg(II) ions.
Related PDB
Related UniProtKB
[11]
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
[12]
Resource
Comments catalysis
Medline ID
PubMed ID 11491304
Journal J Mol Biol
Year 2001
Volume 309
Pages 89-97
Authors Simoncsits A, Tjornhammar ML, Rasko T, Kiss A, Pongor S
Title Covalent joining of the subunits of a homodimeric type II restriction endonuclease: single-chain PvuII endonuclease.
Related PDB
Related UniProtKB
[13]
Resource
Comments catalysis
Medline ID
PubMed ID 11536360
Journal Proteins
Year 2001
Volume 45
Pages 55-61
Authors Dominguez MA Jr, Thornton KC, Melendez MG, Dupureur CM
Title Differential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease.
Related PDB
Related UniProtKB
[14]
Resource
Comments catalysis
Medline ID
PubMed ID 12445784
Journal J Mol Biol
Year 2002
Volume 324
Pages 491-500
Authors Rauch C, Trieb M, Flader W, Wellenzohn B, Winger RH, Mayer E, Hallbrucker A, Liedl KR
Title PvuII-endonuclease induces structural alterations at the scissile phosphate group of its cognate DNA.
Related PDB
Related UniProtKB
[15]
Resource
Comments catalysis
Medline ID
PubMed ID 12475233
Journal Biochemistry
Year 2002
Volume 41
Pages 14848-55
Authors Conlan LH, Dupureur CM
Title Multiple metal ions drive DNA association by PvuII endonuclease.
Related PDB
Related UniProtKB

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.)

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