DB code: S00405

RLCP classification	1.15.9400.1180 : Hydrolysis
CATH domain	3.40.600.20 : ECO RV Endonuclease; Chain A	Catalytic domain
E.C.	3.1.21.4
CSA	1dmu
M-CSA	1dmu
MACiE

CATH domain	Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms
O68557	Type-2 restriction enzyme BglI	R.BglI EC 3.1.21.4 Type II restriction enzyme BglI Endonuclease BglI

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

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
O68557	T2B1_BACSU	Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.			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

1dmuA	Analogue:2x_CA	Bound:A-T-C-G-C-C-T-A-A-T-A-G-G-C-G-A-T (chain F)		Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
Swiss-prot;O68557 & literature [3]

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

1dmuA	LYS 144	ASP 116;ASP 142;ILE 143(two Mg2+ binding)

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[2]	Fig.8, Fig.11, p.12-17	2
[3]	p.5472-5474, Fig.4
[4]	Fig.5, p.13492-13494	2
[5]	Fig.1, p.6

References
[1]
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
[2]
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
[3]
Resource
Comments	X-ray crystallography (2.2 Angstroms)
Medline ID
PubMed ID	9736624
Journal	EMBO J
Year	1998
Volume	17
Pages	5466-76
Authors	Newman M, Lunnen K, Wilson G, Greci J, Schildkraut I, Phillips SE
Title	Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence.
Related PDB	1dmu
Related UniProtKB	O68557
[4]
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
[5]
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

Comments
This enzyme belongs to the type II restriction endonucleases, although it cleaves an interrupted DNA sequence (GCCNNNN/NGGC), unlike other type II restriction endonucleases. According to the paper [2], 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 [2]: (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 [2] 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 [2]. 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 [2]. According to the paper on this enzyme, BglI [3], the active site structure and the way of the dimerization are a bit different from other endonucleases, resulting in different DNA cleavage pattern. For BglI, the metal ions lie along a direction parallel to the scissile phosphodiester bond, whereas for EcoRV, they are positioned on a line approximately perpendicular to it, with Mg2+ ions co-ordinated by Asp74/Asp90 and Glu45/Asp74, respectively [3]. Based on the active site structure, one possible mechanism is that a Mg2+ ion at site 1 could help to activate the attacking water molecule, a Mg2+ at site 2 could help to stabilize the negative charge on the 3' oxyanion leaving group, and both ions could be involved in stabilizing the pentacovalent transition state [3]. The attacking water is also bound to Lys144, which might stabilize the water as in other type II restriction endonucleases (see [3]). ### More recent paper [5] supported the substrate-assisted mechanism for the 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). In addition, the pattern of the active site structure is similar to those of EcoRI, EcoRV, BglI and PvuII (S00403, S00404, S00405, & S00390, respectively in EzCatDB), suggesting a similar catalytic mechanism to those by the enzymes, although the structures with ligand molecules are not available. ### Considering the structure (PDB; 1dmu), which has DNA substrate and 2 calcium ions, and in-line attack by water on the scissile phosphoric ester bond, the substrate-assisted catalytic mechanism is more likely. (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, 2.59 A, and that between the water and calcium ion, 2.83 A). This activated water is stabilized by lys144 (distance 2.84 A). (2) The activated water makes a nucleophilic attack on the phosphorus atom in line with the P-O3' bond. (distance between the water and scissile phosphoric bond, 3.21 A) (3) Transition-state is stabilized by Lys144 and magnesium ion (distance 3.24 A with lys144, and 2.34 A and 2.52 A with the two calcium ions, analogues of magnesium ions). (4) Another water, which is bound to magnesium ion and Asp142, acts as a general acid to protonate the leaving O3' atom (distance between the water and O3' atom, 3.42 A, that between calcium ion and the water, 2.56 A, and that between the water and carboxylate oxygen of Asp142, 2.71 A). (This water also interacts with the phosphate oxygen in the initial state(distance 2.98 A).)

Comments

This enzyme belongs to the type II restriction endonucleases, although it cleaves an interrupted DNA sequence (GCCNNNN/NGGC), unlike other type II restriction endonucleases.
According to the paper [2], 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 [2]:
(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 [2] 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 [2].
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 [2].
According to the paper on this enzyme, BglI [3], the active site structure and the way of the dimerization are a bit different from other endonucleases, resulting in different DNA cleavage pattern.
For BglI, the metal ions lie along a direction parallel to the scissile phosphodiester bond, whereas for EcoRV, they are positioned on a line approximately perpendicular to it, with Mg2+ ions co-ordinated by Asp74/Asp90 and Glu45/Asp74, respectively [3]. Based on the active site structure, one possible mechanism is that a Mg2+ ion at site 1 could help to activate the attacking water molecule, a Mg2+ at site 2 could help to stabilize the negative charge on the 3' oxyanion leaving group, and both ions could be involved in stabilizing the pentacovalent transition state [3]. The attacking water is also bound to Lys144, which might stabilize the water as in other type II restriction endonucleases (see [3]).
###
More recent paper [5] supported the substrate-assisted mechanism for the 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).
In addition, the pattern of the active site structure is similar to those of EcoRI, EcoRV, BglI and PvuII (S00403, S00404, S00405, & S00390, respectively in EzCatDB), suggesting a similar catalytic mechanism to those by the enzymes, although the structures with ligand molecules are not available.
###
Considering the structure (PDB; 1dmu), which has DNA substrate and 2 calcium ions, and in-line attack by water on the scissile phosphoric ester bond, the substrate-assisted catalytic mechanism is more likely.
(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, 2.59 A, and that between the water and calcium ion, 2.83 A). This activated water is stabilized by lys144 (distance 2.84 A).
(2) The activated water makes a nucleophilic attack on the phosphorus atom in line with the P-O3' bond. (distance between the water and scissile phosphoric bond, 3.21 A)
(3) Transition-state is stabilized by Lys144 and magnesium ion (distance 3.24 A with lys144, and 2.34 A and 2.52 A with the two calcium ions, analogues of magnesium ions).
(4) Another water, which is bound to magnesium ion and Asp142, acts as a general acid to protonate the leaving O3' atom (distance between the water and O3' atom, 3.42 A, that between calcium ion and the water, 2.56 A, and that between the water and carboxylate oxygen of Asp142, 2.71 A). (This water also interacts with the phosphate oxygen in the initial state(distance 2.98 A).)

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