EzCatDB: S00405

DB codeS00405
RLCP classification1.15.9400.1180
CATH domainDomain 13.40.600.20Catalytic domain
E.C.3.1.21.4
CSA1dmu


Enzyme Name
Swiss-protKEGG

O68557
Protein nameType-2 restriction enzyme BglItype II site-specific deoxyribonuclease
type II restriction enzyme
SynonymsR.BglI
EC 3.1.21.4
Type II restriction enzyme BglI
Endonuclease BglI


Swiss-prot:Accession NumberO68557
Entry nameT2B1_BACSU
ActivityEndonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.
Subunit
Subcellular location
CofactorBinds 2 magnesium ions per subunit.


CofactorsSubstratesProducts
KEGG-idC00305C00039C00001C00578C00039
CompoundMagnesiumDNAH2ODNA 5'-phosphateDNA
Typedivalent metal (Ca2+, Mg2+)nucleic acidsH2Onucleic acids,phosphate group/phosphate ionnucleic acids
1dmuAAnalogue:2x_CABound:A-T-C-G-C-C-T-A-A-T-A-G-G-C-G-A-T(chain F)
UnboundUnbound

Active-site residues
resource
Swiss-prot;O68557 & literature [3]
pdbCatalytic residuesCofactor-binding residues
1dmuALYS 144
ASP 116;ASP 142;ILE 143(two Mg2+ binding)

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[2]Fig.8, Fig.11, p.12-172
[3]p.5472-5474, Fig.4
[4]Fig.5, p.13492-134942
[5]Fig.1, p.6

references
[1]
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.
[2]
Commentscatalysis
PubMed ID9210460
JournalEur J Biochem
Year1997
Volume246
Pages1-22
AuthorsPingoud A, Jeltsch A
TitleRecognition and cleavage of DNA by type-II restriction endonucleases.
[3]
CommentsX-ray crystallography (2.2 Angstroms)
PubMed ID9736624
JournalEMBO J
Year1998
Volume17
Pages5466-76
AuthorsNewman M, Lunnen K, Wilson G, Greci J, Schildkraut I, Phillips SE
TitleCrystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence.
Related PDB1dmu
Related Swiss-protO68557
[4]
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.
[5]
PubMed ID10739241
JournalProtein Sci
Year2000
Volume9
Pages1-9
AuthorsDall'Acqua W, Carter P
TitleSubstrate-assisted catalysis: molecular basis and biological significance.

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).)

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 2010)
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)

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