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 18420		15377 15377
PubChem	888 888		22247451 962 22247451 962

1eyuA	Unbound	Bound:T-G-A-C-C-A-G-C-T-G-G-T-C (chain D:double stranded DNA)		Unbound	Unbound
1eyuB	Unbound	Bound:T-G-A-C-C-A-G-C-T-G-G-T-C (chain C:double stranded DNA)		Unbound	Unbound
1f0oA	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	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	Unbound	Bound:T-G-A-C-C-A-G-C-T-G-G-T-C (chain D:double stranded DNA)		Unbound	Unbound
1pviB	Unbound	Bound:T-G-A-C-C-A-G-C-T-G-G-T-C (chain C:double stranded DNA)		Unbound	Unbound
1pvuA	Unbound	Unbound		Unbound	Unbound
1pvuB	Unbound	Unbound		Unbound	Unbound
2pviA	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	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	LYS 70	ASP 58;GLU 68(two Mg2+ binding)
1eyuB	LYS 70	ASP 58;GLU 68(two Mg2+ binding)
1f0oA	LYS 70	ASP 58;GLU 68(two Mg2+ binding)
1f0oB	LYS 70	ASP 58;GLU 68(two Mg2+ binding)
1pviA	LYS 70	ASP 58;GLU 68(two Mg2+ binding)
1pviB	LYS 70	ASP 58;GLU 68(two Mg2+ binding)
1pvuA	LYS 70	ASP 58;GLU 68(two Mg2+ binding)
1pvuB	LYS 70	ASP 58;GLU 68(two Mg2+ binding)
2pviA	LYS 70	ASP 58;GLU 68(two Mg2+ binding)
2pviB	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.)

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